Optimization of enzyme replacement therapy for treatment of homocystinuria

ABSTRACT

The present invention provides a method of PEGylating a human truncated cystathionine β-synthase protein containing a mutation of a cysteine to a serine at amino acid position 15 (htCBS C15S). The htCBS C15S was PEGylated with one of 5 kDa, 10 kDa, or 20 kDa NHS ester PEG molecules. In-process monitoring of the PEGylation process was used in the method to reduce levels of unPEGylated htCBS C15S and htCBS C15S with insufficient PEGylation. Administration of the PEGylated htCBS C15S had efficacy throughout the course of treatment for homocystinuria.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 U.S. National Stage Entry of International Application No. PCT/US2018/027854 filed Apr. 17, 2018, entitled “OPTIMIZATION OF ENZYME REPLACEMENT THERAPY FOR TREATMENT OF HOMOCYSTINURIA,” which claims the benefit of priority of U.S. Provisional Patent Application No. 62/486,246, filed Apr. 17, 2017, entitled “OPTIMIZATION OF ENZYME REPLACEMENT THERAPY FOR TREATMENT OF HOMOCYSTINURIA,” the contents of each of which are herein incorporated by reference in their entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled 20891004US371 SL.txt, was created on Oct. 17, 2019, and is 10,892 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to characterizing PEGylated human truncated cystathionine beta-synthase (htCBS) conjugates to identify their suitability for reproducible manufacturing and further clinical development as an enzyme replacement therapy for classical homocystinuria.

BACKGROUND OF THE INVENTION

Human cystathionine beta-synthase (CBS, KEGG enzyme identifier: EC 4.2.1.22) is an important enzyme of the sulfur amino acid metabolism, whose activity determines either recycling of the sulfur in the methionine (Met) cycle or its diversion into synthesis of cysteine through the transsulfuration pathway. See, Finkelstein, J. D., J. Nutr. Biochem. 1990, 1, 228-237, which is hereby incorporated by reference in its entirety. The critical intermediate of sulfur amino acid metabolism is the non-proteinogenic amino acid homocysteine (Hcy), which can be either converted back to Met by an action of Met synthase or betaine homocysteine methyltransferase or redirected by CBS to cysteine biosynthesis via the transsulfuration pathway. CBS is a pyridoxal 5′-phosphate-dependent homotetrameric hemeprotein, which catalyzes the condensation of Hcy with serine forming cystathionine (Cth) and water. Cth is subsequently hydrolyzed by the action of cystathionine gamma-lyase to cysteine (Cys), alpha-ketobutyrate and ammonia, what completes the conversion of the essential amino acid Met to Cys. Activity of CBS, and thus the flux of sulfur through the transsulfuration pathway, is attenuated by the autoinhibitory function of the enzyme's C-terminal regulatory domain. Binding of S-adenosylmethionine to this domain results in a conformational rearrangement of the regulatory domain, subsequent release of the autoinhibitory block and 4-5-fold activation of the enzyme. See, Ereno-Orbea et al., Proc Natl Acad Sci USA 2014, 111, (37), E3845-52, which is hereby incorporated by reference in its entirety. Similar level of CBS activation can be achieved by removal of the C-terminal regulatory domain and consequent formation of a dimeric, permanently activated human truncated enzyme (htCBS). See, Kery et al., Arch Biochem Biophys 1998, 355, (2), 222-32, which is hereby incorporated by reference in its entirety.

Lack of CBS activity results in classical homocystinuria (HCU), an autosomal recessively inherited rare metabolic disorder. See, Mudd et al., Disorders of transsulfuration. In The Metabolic and Molecular Bases of Inherited Disease, 8 ed.; Scriver, C. R.; Beaudet, A. L.; Sly, W. S.; Valle, D.; Childs, B.; Kinzler, K.; Vogelstein, B., Eds. McGraw-Hill: New York, 2001; pp 2007-2056 which is hereby incorporated by reference in its entirety. The inactivation of CBS, chiefly due to the presence of missense pathogenic mutations, yields severe elevation of Hcy, Met, and S-adenosylhomocysteine and conversely lack of Cth and decrease of Cys in plasma samples of HCU patients. These biochemical sequelae are accompanied with connective tissue disorders, such as dislocated optic lenses, long and thin limbs and osteoporosis, thromboembolism, cognitive impairment and other clinical symptoms. See, Mudd et al. Present treatment of HCU is mostly symptomatic aimed at reduction of Hcy concentration in the plasma and tissues by protein-restricted diet with Met-free L-amino acid supplementation. See, Komrower et al., Arch Dis Child 1966, 41, (220), 666-71, which is hereby incorporated by reference in its entirety. This dietary intervention is often combined with betaine, which promotes the remethylation of Hcy back to Met. See, Smolin et al. J Pediatr 99(3), 467-472 (1981), which is hereby incorporated by reference in its entirety. In addition, supplementation with the pyridoxal 5′-phosphate precursor pyridoxine (vitamin B6) was shown to be effective in roughly 50% of HCU patients by stimulating the residual CBS activity and thus relieving the clinical manifestation and relaxing the dietary regime. See, Barber et al. J Pediatr 75(3), 463-778 (1969), which is hereby incorporated by reference in its entirety. Although Met restriction is generally effective in lowering plasma Hcy levels, a compliance of the HCU patients with the diet is quite poor with consequent manifestation of the symptoms. See, Walter et al., Eur. Pediatr. 1998, 157 (Suppl 2), S71-S76, which is hereby incorporated by reference in its entirety. Thus, there is a need in the art for an alternative therapy that would not require a stringent diet but would achieve similar reduction of Hcy levels and prevent onset and/or escalation of the clinical symptoms.

Enzyme replacement therapy (ERT) for HCU has recently been described using PEGylated htCBS. See, Bublil et al., J Clin Invest 2016, 126, (6), 2372-84 and International application PCT/2016/061050, which are hereby incorporated by reference in their entireties. PEGylation is conjugation of protein of interest to one or more polyethylene glycol (PEG) moieties or molecules and is a well-recognized and accepted strategy to alter pharmacokinetic profiles of a variety of drugs to improve their therapeutic potential. See, Fishburn et al., J Pharm Sci 2008, 97, (10), 4167-83, which is hereby incorporated by reference in its entirety. Unmodified htCBS exhibited rapid clearance from the circulation of the mouse model of the disease (elimination half-life after IV administration was 2.7 h). PEGylation of htCBS has been observed to lengthen the elimination half-life of PEGylated htCBS after IV administration by 6-11-fold, improve the plasma metabolic profile of murine homocystinuria in “Human Only” (HO) mice, and rescue the CBS knock-out (KO) mice from neonatal lethality by preventing liver damage. See, Bublil et al., J Clin Invest 2016, 126, (6), 2372-84; each of which is herein incorporated by reference in its entirety.

There is a long-felt need in the art to design and characterize a PEGylated htCBS conjugate that exhibits high reproducibility and control during manufacturing as well as having long-term efficacy. In previous studies, introduction of the C15S point mutation to htCBS was observed to diminish formation of protein aggregates and higher order oligomers and improved PEGylation profile for 20 kDa linear maleimide-activated PEG molecule. See, Bublil et al. Various htCBS C15S conjugate PEGylated with either maleimide (MA) or N-hydroxysuccinimide (NHS) ester-activated PEG molecule (further referred as PEG-htCBS C15S) are characterized herein for their potency to correct metabolic profile of murine homocystinuria after a single administration or multiple rounds of repeated administration interrupted with washout periods. The PEG-htCBS C15S conjugates were ranked based on their pharmacodynamics. In addition, insight was gained into reproducibility and consistency of PEG-htCBS preparation and characterization of the sites modified by the PEG molecules. Assays were identified to monitor and evaluate the extent of htCBS C15S PEGylation.

SUMMARY OF THE INVENTION

Various embodiments of the invention herein provide a method of PEGylating a human truncated cystathionine β-synthase (CBS) protein containing a mutation of a cysteine to a serine at amino acid position 15 of SEQ ID NO: 1 or a variant thereof (htCBS C15S), the method comprising: (a) conjugating the htCBS C15S with one or a plurality of NHS ester PEG molecules in solution at a molar excess of the NHS ester PEG molecules up to about 40-fold to create a batch, wherein the plurality of NHS ester PEG molecules are 5 kDa, 10 kDa, or 20 kDa NHS ester PEG molecules; (b) comparing a retention time from a chromatographic profile from Size Exclusion Chromatography-High Performance Liquid Chromatography (SEC-HPLC) analysis of the batch to a retention time from a chromatographic profile from SEC-HPLC analysis of htCBS C15S with acceptable PEGylation to identify insufficient PEGylation, wherein the batch with insufficient PEGylation has a retention time greater than the retention time of the htCBS C15S with acceptable PEGylation; and (c) adding additional NHS ester PEG molecules to the batch to reduce the amount of insufficient PEGylation in the batch, thereby PEGylating the htCBS C15S.

Various embodiments of the invention herein provide a method of PEGylating a human truncated cystathionine β-synthase (CBS) protein containing a mutation of a cysteine to a serine at amino acid position 15 of SEQ ID NO: 1 or a variant thereof (htCBS C15S), the method comprising: (a) conjugating the htCBS C15S with one or a plurality of NHS ester PEG molecules in solution at a molar excess of the NHS ester PEG molecules less than about 40-fold to create a batch, wherein the plurality of NHS ester PEG molecules are 5 kDa, 10 kDa, or 20 kDa NHS ester PEG molecules; (b) comparing a retention time from non-reduced capillary electrophoresis (NR-CE) analysis of the batch to a retention time from NR-CE analysis of htCBS C15S with acceptable PEGylation to identify insufficient PEGylation, wherein the batch with insufficient PEGylation has a retention time less than the retention time of the htCBS C15S with acceptable PEGylation; and (c) adding additional NHS ester PEG molecules to the batch to reduce the amount of insufficient PEGylation in the batch, thereby PEGylating the htCBS C15S.

In certain embodiments, NHS ester PEG molecules are present in a molar excess of about 10-fold. In certain embodiments, the NHS ester PEG molecules are present in a molar excess of about 20-fold. Alternatively, the NHS ester PEG molecules are present in a molar excess less than 20-fold, less than 10-fold, less than 5-fold, or less than 2-fold. Certain embodiments provide that the plurality of NHS ester PEG molecules consists of about 5 kDa. NHS ester (5NHS) PEG molecules. In certain embodiments, the plurality of NHS ester PEG molecules consists of about 20 kDa NHS ester (20NHS) PEG molecules. In certain embodiments, the plurality of NHS ester PEG molecules is less than about 20 kDa. For example, the plurality of NHS ester PEG molecules consists of about 10 kDa NHS ester (10NHS) PEG molecules. In certain embodiments, htCBS C15S with insufficient PEGylation comprises fewer than 15, fewer than 10, fewer than 5, or fewer than 1 PEGylated amino acids. In certain embodiments, htCBS C15S with acceptable PEGylation comprises at least 1 PEGylated amino acid.

In certain embodiments, the retention time of the batch of htCBS C15S with insufficient PEGylation is greater than about 9.50 minutes, about 9.75 minutes, about 10.00 minutes, and about 10.25 minutes. In certain embodiments, the retention time of the batch of htCBS C15S with insufficient PEGylation is between about 9.60 minutes and about 9.70 minutes. In certain embodiments, the retention time htCBS C15S with acceptable PEGylation is within a range from about 9.50 to about 9.60 minutes. In certain embodiments, the retention time htCBS C15S with acceptable PEGylation is less than about 9.53 minutes. For example, a PEGylated htCBS C15S with insufficient PEGylation comprises zero (0) PEGylated amino acids.

Various embodiments of the invention herein provides a method of maintaining efficacy of a treatment for homocystinuria in a subject throughout the duration of the treatment, the method including the steps of: administering to the subject a therapeutically effective amount of human truncated cystathionine β-synthase (CBS) protein containing a mutation of a cysteine to a serine at amino acid position 15 of SEQ ID NO: 1 or a variant thereof (htCBS C15S) PEGylated using the method above. In certain embodiments, the efficacy of the treatment is maintained for up to 2 weeks, up to 1 month, up to 6 months, or up to 1 year. Alternatively, the efficacy of the treatment is maintained for longer than 1 year.

Various embodiments of the invention herein provide a method of reducing the level of homocysteine (Hcy) in a subject with homocystinuria, the method including: administering to the subject a therapeutically effective amount of human truncated cystathionine β-synthase (CBS) protein containing a mutation of a cysteine to a serine at amino acid position 15 of SEQ ID NO: 1 or a variant thereof (htCBS C15S) PEGylated using the method above. In certain embodiments, the level of Hcy is reduced in a range selected from the group of ranges selected from about 5% to about 10%, about 10% to about 20% about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, and about 90% to about 100%.

Various embodiments of the invention herein provide a method of increasing the level of cysteine (Cys) in a subject with homocystinuria, the method including the steps of: administering to the subject a therapeutically effective amount of human truncated cystathionine 3-synthase (CBS) protein containing a mutation of a cysteine to a serine at amino acid position 15 of SEQ ID NO: 1 or a variant thereof (htCBS C15S) PEGylated using the method above. In certain embodiments, the variant shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1. In certain embodiments, a therapeutic effect resulting from the administering step is maintained for at least 6 hours, at least 12 hours, at least 18 hours, or at least 24 hours.

Various embodiments of the invention herein provide a pharmaceutical composition including a human truncated cystathionine (i-synthase (CBS) protein containing a mutation of a cysteine to a serine at amino acid position 15 of SEQ ID NO: 1 or a variant thereof (htCBS C15S) PEGylated using the method above: and a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the variant in the pharmaceutical composition shares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows non-reduced capillary electrophoresis (NR-CE) profiles of multiple 20NHS PEG-htCBS C15S batches compared to an unmodified htCBS C15S protein.

FIG. 2 shows chromatographic separation of 400MA PEG-htCBS C15S species on DEAE Sepharose using a shallow salt gradient (5%, 10-110 mM NaCl). The straight line represents absorbance at 280 nm, while the peaks show changes in conductivity of the mobile phase. Both lines were generated using BIO-RAD® Biologic LP chromatographic system with BIO-RAD® Biologic LP Data View Software version 1.03. “S” represents the time point of the start of the pump and recording by the software program, and “E” represents the time point of the end of operation of the pump and recording by the software program. “M” represents a manually designated points of the beginning of the monitoring of Pool 1 and Pool 2 as indicated by the labels in the figure.

FIG. 3A-3C show maleimide PEGylation mapping using a Lys-C digest. These figures show reduced alkylated LC-MS peptide maps after a Lys-C digest of unmodified htCBS C15S as a reference (FIG. 3A), homoPEGylated 400MA PEG-htCBS C15S (P1) (FIG. 3B), and hemiPEGylated 400MA PEG-htCBS C15S (P2) (FIG. 3C) proteins.

FIG. 4A-4C show maleimide PEGylation mapping using a NTCB digest. These figures show LC-MS peptide maps after NTCB treatment of unmodified htCBS C15S as a reference (FIG. 4A), homoPEGylated 400MA PEG-htCBS C15S (P) (FIG. 4B), and hemiPEGylated 400MA PEG-htCBS C15S (P2) (FIG. 4C) protein samples.

FIG. 5 shows CBS specific activity of various NHS PEG-htCBS C15S conjugates compared to unmodified htCBS C15S. Bars represent the average from 3 separate measurements and error bars designate standard error of the means (SEMs).

FIG. 6 shows evaluation of 5NHS PEG-htCBS C15S in a washout experiment. Plasma levels of Hcy (squares). Cth (circles) and Cys (diamonds) after repeated administration of 5NHS PEG-htCBS C15S in HO mice (SC, 7.5 mg/kg). Points represent an average value from individual HO mice (n=6) and error bars indicate SEMs.

FIG. 7A-7B show body weight (FIG. 7A) and weight gain (FIG. 7B) of 20NHS PEG-htCBS C15S-treated CBS KO male (solid line and circles) and female (dashed line and circles) mice compared to PBS-injected+/−males (solid line and squares) and females (dashed line and squares). The number of subjects in each group is indicated in the plots. Mice received subcutaneous injections of 7.5 mg/kg 3 times per week from day 2 of age.

FIG. 8A-8B show effects of a long-term treatment of the 1278T mice with 20NHS PEG-htCBS C15S, which sustains improved plasma sulfur amino acid profiles. FIG. 8A shows metabolite concentrations in weaned (day 21) 1278T mice (n=3) that were treated 3× a week with 20NHS PEG-htCBS C15S (SC, 7.5 mg/kg) for about 9 months. Day 21 shows plasma levels of Hcy (solid line and squares), Cth (dashed line and circles), and Cys (dashed line and diamonds) prior the treatment commencement, while days 28 and 63 illustrate the plasma levels 72 hours after the 3rd and 18th injection, respectively (i.e. weekend washout). All subsequent time points show plasma sulfur amino acids 24 hours after dosing. FIG. 8B shows results for the adult, fully symptomatic 1278T mice (n=10) injected 3 times per week with 20NHS PEG-htCBS C15S (SC, 7.5 mg/kg) from age of 26 weeks for a period of about 6 months. Day 182 shows initial levels prior treatment and all the subsequent time points represent plasma metabolites 24 hours after dosing. Symbols represent an average value from individual mice and error bars indicate standard error of the means (SEMs).

FIG. 9A-9C show pharmacokinetics of 20NHS PEG-htCBS C15S after a single dose administration and after a repeated administration to Sprague Dawley rats. FIG. 9A shows a semi-log plot of CBS specific activity in plasma of male (black squares, n=11 each group) and female rats (gray circles, n=8 each group) after a single SC administration of 8 mg/kg (solid lines) or 24 mg/kg 20NHS PEG-htCBS C15S (dashed lines). CBS specific activity in plasma of male (black, n=8) and female rats (gray, n=8) after repeated SC injections of 4 mg/kg (FIG. 9B) or 24 mg/kg 20NHS PEG-htCBS C15S (FIG. 9C). The gray arrows designate a dosing time point for a total of 9 doses administered every 2 days. Data points in all plots represent average values and error bars show SEMs.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Homocystinuna (HCU) due to loss of CBS causes accumulation of homocysteine (Hcy) and depletion of cysteine (Cys). There is a long felt need in the art for an optimized targeted treatment, which would address the underlying enzyme deficiency, thus the development of an enzyme replacement therapy based on PEGylated human truncated CBS (PEG-htCBS) has been explored herein. Attenuation of potency was observed, which warranted screening of several different PEG-htCBS C15S conjugates. The PEG-htCBS C15S conjugates were ranked based on their efficacy to correct plasma metabolite profile of murine homocystinuria after repeated administrations interrupted with washouts. CBS was observed to couple with maleimide PEG molecules inconsistently. In contrast, the PEG-htCBS C15S conjugate with 20 kDa linear NHS-PEG showed very little loss of potency likely due to reproducible PEGylation resulting in species modified with an average of 5 PEGylation sites per peptide. In addition, assays were developed to monitor the extent of CBS PEGylation. These results identified the PEG-htCBS C15S conjugate of those analyzed most suitable for manufacturing and clinical development.

An integral part of any novel therapeutic development effort, whether it is a small molecule drug or a biologic, is the selection, optimization and characterization of a lead candidate to enter the clinical trials. Although effective, treatment of HCU to reduce methionine (Met) intake through dietary restrictions is very difficult to follow and results in serious patient compliancy issues. See, Walter et al., Eur. J. Pediatr. 1998, 157 (Suppl 2), S71-S76, which is hereby incorporated by reference in its entirety. The therapeutic goal of the currently available treatments for HCU, i.e., a restricted diet, pyridoxine, and/or betaine supplementation, is to reduce or normalize plasma Hcy levels because fluctuations and high levels of Hcy are often associated with serious complications, particularly thromboembolic events. See, Morris et al., Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency. J Inherit Metab Dis 2016, which is hereby incorporated by reference in its entirety. Development of an alternative treatment, such as ERT, would reduce or eliminate reliance on dietary restrictions, which would allow HCU patients and their families access to a better quality of life. To achieve this therapeutic goal (i.e. the reduction or normalization of plasma levels of Hcy), the htCBS C15S variant has been previously modified with 20 kDa linear maleimide-activated PEG molecule and proof of concept was provided using murine models of HCU. See, Bublil et al., J Clin Invest 2016, 126, (6), 2372-84 and International application PCT/2016/061050, which are hereby incorporated by reference in their entireties.

There is a trend in PEGylation of therapeutic macromolecules from heterogeneous mixtures of conjugate isomers towards a single modified form, such as characterization/identification of the product and reproducibility of the manufacturing process. See, Pasut et al., J Control Release 2012, 161, (2), 461-72, which is hereby incorporated by reference in its entirety. Modification of accessible Cys residues using maleimide PEG molecules is one of the most commonly used approaches for site selective PEGylation. Despite the mutagenesis of the most accessible cysteine residue 15 into serine (C15S) in htCBS, multiple PEGylated species were observed in addition to a fraction of an unmodified enzyme. See, Frank et al., Biochemistry 2006, 45, (36), 11021-9, which is hereby incorporated by reference in its entirety. Although the individual species were chromatographically separable, such operation would certainly result in substantial losses and/or would likely render the process economically non-viable. Maleimide PEGylation of htCBS C15S was mapped to a peptide containing residues C272 and C275 with the former residue being a primary site for modification. Mutagenesis of either Cys residue would most likely increased reproducibility of PEGylation; however, substantial reduction in heme saturation and catalytic activity was anticipated. See, Taoka et al., Biochemistry 2002, 41, (33), 10454-61, which is hereby incorporated by reference in its entirety.

Similar strategy for PEGylation was employed in the development of an ERT for phenylketonuria based on phenylalanine ammonia-lyase (PAL). Unlike CBS described herein, the PAL is a tetrameric non-mammalian enzyme originating from cyanobacterial and fungal species. See, Sarkissian et al., Proc Natl Acad Sci USA 2008, 105. (52), 20894-9, which is hereby incorporated by reference in its entirety. Therefore, immunogenicity of such a foreign protein represented a substantial concern and various PEGylation protocols were tested with PAL. See, Gamez et al., Molecular therapy: the journal of the American Society of Gene Therapy 2005, 11, (6), 986-9, which is hereby incorporated by reference in its entirety. PAL was conjugated with NHS ester PEGs ranging from a linear 5 kDa up to a branched 40 kDa in size with linear 20 kDa PEG-PAL conjugates demonstrating increased performance and masking immunogenicity the best. Reduction of the PEG-PAL immunogenicity was directly proportional to the number of PEGs attached to the PAL subunit with gel-based estimate of 6-8 PEGs per PAL monomer suppressing the immune response most efficiently. See, Gamez et al.

When PEG-PAL was administered to the PKU mouse model, plasma phenylalanine levels were brought down to zero in short-term study and after overcoming an initial immune response also in a long-term study. See, Sarkissian et al., Proc Natl Acad Sci USA 2008, 105, (52), 20894-9, which is hereby incorporated by reference in its entirety. The Hcy metabolism and pathophysiology need to be taken into an account. Unlike phenylalanine, Hcy is a redox-active thiol group-containing molecule existing in plasma in a variety of chemical forms (as fractions of total Hcy pool): thiolactone (<1%), free reduced (about 2%0/), disulfide (about 18%), and protein-bound (about 80%). Refsum et al., Clin Chem 1985, 31, 624-628; Rasmussen et al. Ann Clin Biochem 2000, 37 (Pt 5), 627-48; Jakubowski. H., Anal Biochem 2002, 308, (1), 112-9, each of which are hereby incorporated by reference in their entireties.

Free Hcy is increased substantially to a 10-25% of total plasma Hcy in HCU. See, Mansoor et al., Metabolism 1993, 42, 1481-1485. In addition, Hcy, in homocystinuria, substantially replaces Cys bound to plasma albumin, what may contribute to decreased levels of plasma Cys found in HCU. See, Bar-Or et al., Biochem Biophys Res Commun 2004, 325, (4), 1449-53, which is hereby incorporated by reference in its entirety. Albumin is synthesized exclusively in the liver and its plasma concentration of 33-52 g/l in healthy human represents only about 40%, while the remaining about 60% of albumin stays in extravascular space. See, Boldt et al., Br J Anaesth 2010, 104, (3), 276-84, which is hereby incorporated by reference in its entirety. In theory, only a free Hcy represents a substrate for CBS. Thus, the 50-80% decrease of total Hcy in HO mice upon continuous administration of 20NHS PEG-htCBS C15S may be explained by shifting the balance of metabolites in favor of free Hcy compared to other forms (disulfides and protein-bound). See, Bublil et al., J Clin Invest 2016, 126, (6), 2372-84 and International application PCT/2016/061050, which are hereby incorporated by reference in their entireties. 20NHS PEG-htCBS C15S in circulation serves as a “sink” for excess of Hcy to improve the Hcy plasma levels. Possible reasons for failing to normalize levels could be the constant flux of intracellular Hcy (particularly from the liver as Hcy bound to a newly synthesized albumin), dynamic balance between intra- and extra-vascular Hcy pools (mediated mostly by albumin), and homocystinuria-induced pro-oxidative environment favoring disulfides and protein-bound forms of total Hcy pool. Nevertheless, recently published guidelines for diagnosis and management of HCU recommend keeping the plasma total of Hcy below 100 μM to prevent progress and/or occurrence of serious complications. See, Morris et al., Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency. J Inherit Metab Dis 2016, which is hereby incorporated by reference in its entirety. Values are extrapolated from the results herein from homocystinuric HO mice fed with standard unrestricted chow containing 4-5 g Met per kg of diet (corresponds to 18-19% of protein). Administration of 20NHS PEG-htCBS C15S to human HCU patients results in guideline-recommended total Hcy levels in plasma without any additional treatment. Based on the results herein, a GLP-toxicology (GLP-tox) study in humans using 20NHS PEG-htCBS C15S as an ERT for HCU has been initiated.

II. Methods of Characterizing Pegylated HtCBS C15S

A. Extent of PEGylation of htCBS C15S (PEG-htCBS C15S)

In certain embodiments, the extent of PEGylation of htCBS C15S may be used to define acceptance criteria for individual PEG-htCBS C15S. In some embodiments, the PEG molecule is ME-200GS (also know n as 20NHS).

In certain embodiments, size exclusion chromatography (SEC)-high performance liquid chromatography (HPLC) is used to compare a batch of PEG-htCBS C15S during processing to unmodified htCBS C15S. Insufficiently PEGylated htCBS batches are identified by longer retention times than the retention times for acceptable PEGylation. In certain embodiments, a retention time of greater than about 9.5 minutes, about 9.75 minutes, about 10 minutes, and about 10.25 minutes indicates insufficient PEGylation (<1 PEG/protein or <5 PEGs/protein). For example, a retention time between 9.6 and 9.7 minutes, such as 9.67 minutes, may indicate insufficient PEGylation.

In certain embodiments, a retention time of less than about 9.6 minutes, about 9.4 minutes, about 9.2 minutes, and about 9 minutes was determined to indicate acceptable PEGylation (at least 1 PEG/protein). For example, a retention time between 9.5 and 9.6 minutes, such as 9.53 minutes, may indicate acceptable PEGylation.

In some embodiments, additional PEG molecules may be added to the reaction mixture for insufficiently PEGylated batches to potentially rescue the extent of PEGylation. The comparison by SEC-HPLC may be performed multiple times during the PEGylation process. Alternatively, the comparison by SEC-HPLC may be performed once during the PEGylation process.

In certain embodiments, non-capillary electrophoresis is used to quantify multiple PEG-htCBS C15S species. Insufficiently PEGylated batches of PEG-htCBS C15S were identified by the presence of unmodified enzyme and an increased occurrence of low molecular weight PEG-htCBS C15S species. In certain embodiments, liquid chromatography-mass spectrometry (LC-MS) is used to estimate the number of PEG molecules per peptide. In certain embodiments, at least seven PEGylated lysines are observed to be modified in an PEGylated htCBS C15S with acceptable PEGylation. For example, K25, K30, K211, K247, K271, K405, and K406 of htCBS C15S may be PEGylated. The lysines may have varying degrees of PEGylation. More specifically, K25 and K30 of htCBS C15S may be PEGylated to the full extent, while other lysines may be partially PEGylated.

In certain embodiments, acceptable PEGylation is defined as about 5 PEGylated sites per htCBS C15S protein. Alternatively, acceptable PEGylation may be selected from at least one range of between 1-5 PEGylated sites, between 3-7 PEGylated sites, and between 5-9 PEGylated sites per htCBS CBS protein. For example, acceptable PEGylation of htCBS C15S may include one PEGylated lysine per protein. Alternatively, acceptable PEGylation of htCBS C15S may include about 5 PEGylated lysines per protein.

B. Optimizing PEG Conjugation

Various approaches such as reduction of accessible cysteines with tris(2-carboxyethyl)phosphine (TCEP) prior to PEGylation to enhance the yield of the modified enzyme or multiple PEGylation schemes to gain consistency in the process have been previously observed to partially improve the consistency of PEGylation.

Maleimide PEGylation of htCBS may be site-specific. In certain embodiments, htCBS C15S is first PEGylated at C272 during maleimide PEGylation. Alternatively, htCBS C15S is PEGylated at C272 and C275 during maleimide PEGylation.

Alternatively, htCBS C15S may be PEGylated by at least one of 5, 10, or 20 kDa linear NHS ester PEG molecules. In certain embodiments, PEGylation of htCBS C15S by a NHS ester PEG molecule results in a reproducible pattern. In certain embodiments, PEGylation is performed with up to 20-fold excess of PEG molecule to the CBS protein. For example, PEGylation of htCBS C15S is performed with between about 10-fold and 20-fold excess of 5 kDa NHS ester PEG (5NHS), 10 kDa NHS ester PEG (10NHS), or 20 kDa NHS ester PEG (20NHS) molecules.

Alternatively, PEGylation is performed with about 40-fold to about 60-fold excess of PEG molecule to the CBS protein. In one aspect, PEGylation of htCBS C15S is performed with about 40-fold excess of 5 kDa NHS ester PEG (5NHS), 10 kDa NHS ester PEG (10NHS), or 20 kDa NHS ester PEG (20NHS) molecules. Alternatively. PEGylation of htCBS C15S is performed with less than about 40-fold excess of 5 kDa NHS ester PEG (5NHS), 10 kDa NHS ester PEG (10NHS), or 20 kDa NHS ester PEG (20NHS) molecules.

In certain embodiments, residual ammonium sulfate from chromatographic purification is removed from an unmodified batch of CBS protein, such as unmodified htCBS C15S, to increase reproducibility of the PEGylation pattern as compared to the reproducibility of the PEGylation pattern of htCBS C15S in a mixture containing the residual ammonium sulfate. For example, the ammonium sulfate may be removed using a buffer exchange technique.

Following PEGylation, unreacted PEG molecules are removed from the PEGylation mixture to reduce its viscosity by increasing the surface area of the diafiltration membrane. For example, the surface area is increased by about 2-fold, about 3-fold, or about 4-fold. Alternatively, viscosity may be reduced by diluting the PEGylation mixture. For example, the PEGylation mixture is diluted by about 2-fold. In certain embodiments, the unreacted PEG molecules or unmodified htCBS C15S is removed from the mixture to reduce immunogenicity.

III. Treatment with PEGylated HtCBS C15S

In certain embodiments, a mammal may be administered htCBS C15S PEGylated with a 5 kDa ester NHS PEG molecule or a 20 kDa ester NHS PEG molecule. For example, 5NHS PEG-htCBS C15S or 20NHS PEG-htCBS C15S. Administration of 5NHS PEG-htCBS C15S or 20NHS PEG-htCBS C15S reduces levels of Hcy in plasma or in kidney, liver, or brain tissue by at least 40%. For example, Hcy levels are reduced by more than 40%, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%. Administration of 5NHS PEG-htCBS C15S or 20NHS PEG-htCBS C15S increases levels of Cth in plasma or in kidney, liver, or brain tissue by at least 70%. For example, Cth levels are increased by at least 80%, at least 90%, or at least 99%. Administration of 5NHS PEG-htCBS C15S or 20NHS PEG-htCBS C15S increases levels of Cys in plasma or in kidney, liver, or brain tissue by at least 30%. For example, Cys levels are increased by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%.

Administration of 20NHS PEG-htCBS C15S may reduce levels of homolanthionine (Hlth) in plasma or in kidney, liver, or brain tissue by at least 50%. For example, Hlth levels are reduced by at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%.

The therapeutic effects of administration of 20NHS PEG-htCBS C15S may continue for at least 12 hours of at least 24 hours, at least 48 hours, or at least 72 hours. Specifically, the therapeutic effects may continue for about 24 hours.

S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH) are metabolites involved in the conversion of methionine to homocysteine during the proximal portion of the methionine cycle. See, Krijt, et al. J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Jul. 15: 877(22-3): 2061-2066, which is hereby incorporated by reference herein in its entirety. In certain embodiments, homocystinuria results in reduction of the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) (SAM/SAH), which indicates a decrease in methylation capacity. In certain embodiments, the SAM/SAH ratio may be increased or normalized by administration of a PEG-htCBS C15S to a subject. For example, administration of a 20NHS PEG-htCBS C15S conjugate reduces SAH accumulation, thus increasing the SAM/SAH ratio, thereby normalizing the balance of metabolites in a subject.

IV. Dosing and Administration

In certain embodiments, PEG-htCBS C15S may be administered to a subject by parenteral administration.

In certain embodiments, PEG-htCBS C15S may be administered to a subject by subcutaneous (SC), intravenous (IV) or intraperitoneal (IP) injection. As a non-limiting example, PEG-htCBS C15S may be administered to a subject by subcutaneous administration. As a non-limiting example, PEG-htCBS C15S may be administered to a subject by intravenous administration. As a non-limiting example, PEG-htCBS C15S may be administered to a subject by intraperitoneal administration.

In certain embodiments, PEG-htCBS C15S may be administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 times.

In certain embodiments, the administration of the PEG-htCBS C15S may be repeated every minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, six 6, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, hourly, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, daily, 2 days, 3 days, 4 days, 5 days, 6 days, weekly, biweekly, 3 weeks, monthly, 2 months, quarterly, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, yearly, 13 months, 14 months, 15 months, 16 months, 17 months, or 18 months.

In certain embodiments, the administration of the PEG-htCBS C15S may be a series of doses which are minutes, hours, days or weeks apart. The number of doses in a series may be 2, 3, 4, 5 or 6. As a non-limiting example, a subject is administered 3 doses 24 hours apart. As another non-limiting example, a subject is administered 5 doses 12 hours apart.

In certain embodiments, the administration of the PEG-htCBS C15S may follow a dosing schedule of a series of doses that has a gap between the first series and the second series of doses. The gap between the doses may be 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, day, 2 days, 3 days, 4 days, 5 days, 6 days, week, 2 weeks, 3 weeks, monthly, 2 months, quarterly, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, or 18 months. The number of doses in a series may be 2, 3, 4, 5 or 6. As a non-limiting example, a subject may be administered a first series of 5 doses 12 hours apart and then 14 days after the first dose a subject is administered a second series of 5 doses 12 hours apart. As another non-limiting example, a subject is administered two series of doses over a period of 8 weeks where the first series is one dose twice a week for two weeks and the second series of doses is three times a week for 6 weeks.

In one embodiment, PEG-htCBS C15S may be administered at least once after a subject has been administered betaine. The time between the betaine administration the PEG-htCBS C15S may be 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, day, 2 days, 3 days, 4 days, 5 days, 6 days, week, 2 weeks, 3 weeks, monthly, 2 months, quarterly, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, or 18 months. As a non-limiting example, the PEG-htCBS C15S may be administered 14 days after the subject was administered betaine. As another non-limiting example, a subject may be administered two doses after the subject was administered betaine. The PEG-htCBS C15S mutant may be administered 14 and 15 days after betaine administration.

In one embodiment, PEG-htCBS C15S mutant may be administered in combination with betaine to a subject. The combination may be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 times.

In one embodiment. PEG-htCBS C15S mutant may be administered in combination with betaine to a subject after the subject has initially received betaine. The time between the combination treatment and the original betaine administration may be 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, day, 2 days, 3 days, 4 days, 5 days, 6 days, week, 2 weeks, 3 weeks, monthly, 2 months, quarterly, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, or 18 months. As a non-limiting example, the combination may be administered 14 days after the subject was first administered betaine. As another non-limiting example, a subject may be administered two doses after the subject was first administered betaine. The combination may be administered 14 and 15 days after betaine administration.

In one embodiment, the dose of PEG-htCBS C15S mutant administered to a subject may be between 0.01 mg/kg and 1 mg/kg such as 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg or 1 mg/kg, or between 5 and 8 mg/kg such as 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg or 8 mg/kg. Alternatively, the dose of PEG-htCBS C15S mutant administered to a subject may be between about 2 mg/kg and about 8 mg/kg, about 4 mg/kg and about 16 mg/kg, about 6 mg/kg and 24 mg/kg. For example, the dose is about 24 mg/kg or about 8 mg/kg.

In one embodiment, the PEG-htCBS C15S may be co-administered with another therapeutic for treating CBSDH. As used herein, “co-administered” means the administration of two or more components. Components for co-administration include, but are not limited to PEG-htCBS C15S, betaine or Vitamin B6. Co-administration refers to the administration of two or more components simultaneously or with a time lapse between administration such as 1 second, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 1.5 days, 2 days, or more than 3 days.

V. Definitions

As used in this specification, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “polymer” includes a single polymer as well as two or more of the same or different polymers, or a reference to an “excipient” includes a single excipient as well as two or more of the same or different excipients, and the like.

Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure and specifically disclosed. For example, if a range of 1 μm to 8 μm is stated, it is intended that 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μm are also explicitly disclosed, as well as the range of values greater than or equal to 1 μm and the range of values less than or equal to 8 μm.

“Recombinant,” when used to reference, e.g., a cell, nucleic acid, polypeptide, expression cassette or vector, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified by the introduction of a new moiety or alteration of an existing moiety, or is identical thereto but produced or derived from synthetic materials. For example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell (i.e., “exogenous nucleic acids”) or express native genes that are otherwise expressed at a different level, typically, under-expressed or not expressed at all.

Recombinant techniques can include, e.g., use of a recombinant nucleic acid, such as a cDNA encoding a protein or an antisense sequence, for insertion into an expression system, such as an expression vector: the resultant construct is introduced into a cell, and the cell expresses the nucleic acid, and the protein, if appropriate. Recombinant techniques also encompass the ligation of nucleic acids to coding or promoter sequences from different sources into one expression cassette or vector for expression of a fusion protein, constitutive expression of a protein, or inducible expression of a protein.

The terms “subject”, “individual” or “patient” are used interchangeably herein and refer to a vertebrate, preferably a mammal. Mammals include, but are not limited to, humans.

“Associated” refers to coincidence with the development or manifestation of a disease, condition or phenotype. Association may be due to, but is not limited to, genes responsible for housekeeping functions whose alteration can provide the foundation for a variety of diseases and conditions, those that are part of a pathway that is involved in a specific disease, condition or phenotype and those that indirectly contribute to the manifestation of a disease, condition or phenotype.

“Physiological conditions” or “physiological solution” refers to an aqueous environment having an ionic strength, pH, and temperature substantially similar to conditions in an intact mammalian cell or in a tissue space or organ of a living mammal. Typically, physiological conditions comprise an aqueous solution having about 150 mM NaCl, pH 6.5-7.6, and a temperature of approximately 22-37° C. Generally, physiological conditions are suitable binding conditions for intermolecular association of biological macromolecules. For example, physiological conditions of 150 mM NaCl, pH 7.4, at 37° C. are generally suitable.

“Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient. The term refers to an excipient that can be taken into the mammalian subject's body in association with an active compound (e.g. PEG-htCBS C15S) with no significant adverse toxicological effects to the subject.

The term “excipient” or “vehicle” as used herein means any substance, not itself a therapeutic agent, used as a carrier for delivery of a therapeutic agent and suitable for administration to a subject, e.g. a mammal or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition into a discrete article such as a capsule or tablet suitable for oral administration. Excipients and vehicles include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is nontoxic, and which does not interact with other components of the composition in a deleterious manner. Administration can mean oral administration, inhalation, enteral administration, feeding or inoculation by intravenous injection. The excipients may include standard pharmaceutical excipients and may also include any components that may be used to prepare foods and beverages for human and/or animal consumption, feed or bait formulations or other foodstuffs.

“Permeant”, “drug”, and “pharmacologically active agent” or any other similar term means any chemical or biological material or compound, inclusive of peptides, suitable for administration by the methods previously known in the art and/or by the methods taught in the present disclosure, that induces a desired biological or pharmacological effect, which may include, but is not limited to (1) having a prophylactic effect on the organism and preventing an undesired biological effect such as preventing an infection, (2) alleviating a condition caused by a disease, for example, alleviating pain or inflammation caused as a result of disease, and/or (3) either alleviating, reducing, or completely eliminating the disease from the organism. The effect may be local, such as providing for a local anesthetic effect, or it may be systemic. This disclosure is not drawn to novel permeants or to new classes of active agents. Rather it is limited to the mode of delivery of agents or permeants which exist in the state of the art or which may later be established as active agents and which are suitable for delivery by the present disclosure.

The term “about”, particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

“Optional” or “optionally” means that the subsequently described circumstance may or may not be present or occur, so that the description includes instances where the circumstance is present or occurs and instances where it is not present or does not occur.

“Substantially absent” or “substantially free” of a certain feature or entity means nearly totally or completely absent the feature or entity. For example, for a subject administered PEG-htCBS C15S, the substantial absence of an observable side effect means that such side effect is either non-detectable, or occurs only to a negligible degree, e.g., to an extent or frequency that is reduced by about 50% or more when compared to either the frequency or intensity of the same side effect observed in an untreated patient.

The terms “pharmacologically effective amount” or “therapeutically effective amount” as related to the present composition refer to a non-toxic, but sufficient amount of the active agent (or composition containing the active agent) to provide the desired level in the bloodstream or at the site of action (e.g. intracellularly) in the subject to be treated, and/or to provide a desired physiological, biophysical, biochemical, pharmacological or therapeutic response, such as amelioration of the manifestations of homocystinuria. The exact amount required will vary from subject to subject, and will depend on numerous factors, such as the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (i.e., the number of doses administered per day), as well as patient considerations, such as species, age, and general condition of the subject, the severity of the condition being treated, additional drugs being taken by the subject, mode of administration, and the like. These factors and considerations can readily be determined by one skilled in the art, based upon the information provided herein. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

The term “biological activity” refers to any biological activity typically attributed to a nucleic acid or protein by those skilled in the art. Examples of biological activities are enzymatic activity, ability to dimerize, fold or bind another protein or nucleic acid molecule, etc.

The term “nucleic acid” may be in the form of RNA or in the form of DNA, and include messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded may be the coding strand or the non-coding (anti-sense, complementary) strand.

As used herein, a “variant” is a nucleic acid, protein or polypeptide which is not identical to, but has significant homology (for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) over the entire length of the wild type nucleic acid or amino acid sequence, as exemplified by sequences in the public sequence databases, such as GenBank. As used herein, a “protein, polypeptide or peptide fragment thereof” means the full-length protein or a portion of it having an amino acid sequence usually at least 5, 6, 7, 8. 9, 10, 11, 12, 13, 14 or 15 amino acids in length, although dipeptides, tripeptides and tetrapeptides are also contemplated and encompassed by the present disclosure.

As used herein, a “mutant” is a mutated protein designed or engineered to alter properties or functions relating to glycosylation, protein stabilization and % or ligand binding.

As used herein, the terms “native” or “wild-type” relative to a given cell, polypeptide, nucleic acid, trait or phenotype, refers to the form in which that is typically found in nature.

As used herein, the terms “protein”, “polypeptide”, “oligopeptide”, and “peptide” have their conventional meaning and are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). Furthermore, the polypeptides described herein are not limited to a specific length. Included within this definition are D- and L-amino acids, and mixtures ofD- and L-amino acids. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Polypeptides can also refer to amino acid subsequences comprising epitopes, i.e., antigenic determinants substantially responsible for the immunogenic properties of a polypeptide and being capable of evoking an immune response.

“Position corresponding to” refers to a position of interest (i.e., base number or residue number) in a nucleic acid molecule or protein relative to the position in another reference nucleic acid molecule or protein. Corresponding positions can be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, for example, such that identity between the sequences is greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99%. The position of interest is then given the number assigned in the reference nucleic acid molecule. For example, if a particular polymorphism in Gene-X occurs at nucleotide 2073 of SEQ ID No. X, to identify the corresponding nucleotide in another allele or isolate, the sequences are aligned and then the position that lines up with 2073 is identified. Since various alleles may be of different length, the position designated 2073 may not be nucleotide 2073, but instead is at a position that “corresponds” to the position in the reference sequence.

“Percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted. e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol, 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length “W” in the query sequence, which either match or satisfy some positive-valued threshold score “T” when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as “seeds” for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues: always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments: or the end of either sequence is reached. The BLAST algorithm parameters W, T. and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff& Henikoff. Proc. Natl. Acad Sci. USA 89:10915 (1989)).

While all the above-mentioned algorithms and programs are suitable for a determination of sequence alignment and % sequence identity, for purposes of the disclosure herein, determination of % sequence identity will typically be performed using the BESTFIT or GAP programs in the GCG Wisconsin Software package (ACCELRYS®, Madison Wis.), using default parameters provided.

The term “L or D form amino acids” may be used in relation to the peptide described herein. As will be recognized by skilled artisans, the various X^(n) residues comprising the compounds of the invention may be in either the L- or D-configuration about their C_(α) carbons. In one embodiment, all the C_(α) carbons of a particular compound are in the same configuration. In some embodiments of the invention, the compounds comprise specific chiralities about one or more C_(α) carbon(s) and/or include non-peptide linkages at specified locations to impart the compound with specified properties. For example, it is well-known that peptides composed in whole or in part of D-amino acids are more resistant to proteases than their corresponding L-peptide counterparts. Thus, in one embodiment, the compounds are peptides composed in whole or in part of D-amino acids. Alternatively, compounds having good stability against proteases may include peptide analogs including peptide linkages of reversed polarity at specified positions. For example, compounds having stability against tryptic-like proteases include peptide analogs having peptide linkages of reversed polarity before each L-Arg or L-Lys residue: compounds having stability against chymotrypsin-like proteases include peptide analogs having peptide linkages of reversed polarity before each small and medium-sized L-aliphatic residue or L-non-polar residue. In another embodiment, compounds having stability against proteases include peptide analogs composed wholly of peptide bonds of reversed polarity. Other embodiments having stability against proteases will be apparent to those of skill in the art. Additional specific embodiments of the compounds are described herein.

Described herein are compositions, methods, processes, kits and devices for the design, preparation, manufacture and/or formulation of PEG-htCBS C15S proteins. The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects and advantages of the invention will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present description will control.

The present invention is further illustrated by the following non-limiting examples.

VI. Examples Example 1. Experimental Procedures

A. Chemicals

Unless stated otherwise, all materials were purchased from SIGMA-ALDRICH® or FISHER SCIENTIFIC™. L-[¹⁴C]-serine was obtained from PERKIN ELMER®, Life Sciences.

B. Animals

All animal procedures were approved under animal protocol #B-49414(03)1E by the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC), which is an AAALAC INTERNATIONAL®-accredited (#00235), Public Health Service-assured (#A 3269-01) and USDA-licensed (#84-R-0059) institution. “Human Only” (HO) mice were previously generated and propagated and genotyped as described in Maclean et al., Mol. Genet. Metab. 2010, 101, (2-3), 153-62; Bublil et al., J Clin Invest 2016, 126, (6), 2372-84: and International application PCT/2016/061050, which are hereby incorporated by reference in their entireties. Animals were maintained on extruded standard diet 2918, 2919, or 2920X from TEKLAD GLOBAL RODENT DIETS® (Harlan. Livermore, Calif., USA). A single-use lancet for submandibular bleeding was used for blood collection into CAPIJECT®, T-MLHG lithium heparin (12.5 IU) tubes with gel (Terumo Medical Corporation, NJ, USA). Tubes were then centrifuged at 1,200×g for 10 minutes, followed by transfer of plasma to 1.5 ml tubes and storage at −80° C.

C. Determination of Metabolite Concentrations

Plasma metabolites were determined by stable-isotope-dilution gas chromatography mass spectrometry as previously described. See, Allen et al., Metabolism 1993, 42, 1448-1460. The analysis was performed in a blinded fashion without knowing the animal genotype and/or treatment regimen.

D. Size Exclusion Chromatography-High Performance Liquid Chromatography (SEC-HPLC)

CBS and PEG-htCBS C15S preparations were separated on BioSEC-5 7.8×300 mm, 1000 Å pore size column (Agilent, CA, USA). Column was equilibrated and operated in 100 mM sodium phosphate, pH 6.8, 150 mM NaCl at a flow rate of 1 ml/min at room temperature. Protein samples diluted in mobile phase to a final concentration of 2 mg/ml were loaded on a column via 10 μl sample loop and detected using UV detector at wavelength of 225 nm. Chromatograms were recorded and analyzed using 32 karat software (BECKMAN COULTER®), CA, USA).

E. Non-Reduced Capillary Electrophoresis (NR-CE)

CBS and PEG-htCBS C15S preparations were analyzed on PA800 capillary electrophoresis system (BECKMAN COULTER®, CA. USA) equipped with 30 cm long, 50 μm ID bare-fused silica capillary. The capillary was cut to provide a total length of 30 cm with a distance of approximately 20 cm from sample introduction inlet to detection window and installed in a cartridge with a 100×800 μm aperture. The capillary was preconditioned with 0, 1 M NaOH wash followed by 0.1 M HCl wash before rinsing it with water and final equilibration with SDS MW sample buffer. Protein samples of concentrations less than 10 mg/ml and in a formulation containing high salt were buffer exchanged in water twice (about 7-fold dilution each time) to remove matrix interference, diluted to 1 mg/ml final concentration, and denatured by heating at 80° C. for 5 minutes. The sample was loaded by a high-resolution method using electrokinetic injection (5 kV) for a duration of 20 seconds and separated for 50 minutes under voltage of 15 kV. Electropherograms were recorded at a UV wavelength of 220 nm and analyzed using 32 karat software (BECKMAN COULTER®, CA, USA).

F. Protein Purification and PEGylation

Human truncated CBS carrying the C15S mutation (htCBS C15S) was expressed and purified as described in Bublil et al., J Clin Invest 2016, 126, (6), 2372-84, International application PCT/2016/061050, which are hereby incorporated by reference in their entireties. The purified enzyme was formulated and concentrated using the LABSCALE™ Tangential Flow Filter (TFF) system (EMD Millipore, MA, USA) equipped with PELLICON®, XL 50 ULTRAcel 30 cartridge (regenerated cellulose 30 kDa molecular weight cut-off (MWCO) membrane) into 200 mM potassium phosphate pH 6.5 or 100 mM sodium phosphate pH 7.2. Activated PEG molecules with either a maleimide or a NHS ester coupling group were purchased from NOF Corporation (Japan). PEGylation of htCBS C15S with maleimide and NHS ester PEG molecules was carried out in 100 mM potassium phosphate pH 6.5 and 50 mM sodium phosphate pH 7.2, respectively, by adding PEG molecules dissolved in MILLIQ®, purified water in the desired molar ratio (typically CBS protein:PEG=1:10) to a final protein concentration of 5 mg/ml. Reaction was carried out at 4° C. overnight. Subsequently, reaction mixture was diluted twice with MILLIQ® purified water, buffer exchanged into Gibco 1×PBS (Thermo Fisher Scientific, MA. USA) and concentrated using LABSCALE™ TFF system with PELLICON® XL 50 Biomax 100 cartridge (polyether sulfone 100 kDa MWCO membrane). The final PEG-htCBS C15S conjugates were filter sterilized (Millipore's 0.2 μm PVDF membrane filter), aliquoted, flash frozen in liquid nitrogen and stored at −80° C.

G. Protein Gel Electrophoresis

Protein concentrations were determined by the Bradford assay (Thermo Pierce, MA, USA) using bovine serum albumin (BSA) as a standard per the manufacturer's recommendations. Denatured proteins were separated by SDS-PAGE using a 10% Mini-PROTEAN®. Tris-glycine extended (TGX) gels (BIORAD®, CA, USA), while native samples were resolved by native PAGE in 4-15% Mini-PROTEAN® TGX gels. For visualization, the gels were stained with Simple Blue (Invitrogen, CA, USA). Free PEG molecules and PEGylated htCBS C15S were visualized using barium iodide staining as described in Kurfurst et al., Anal Biochem 1992, 200, (2), 244-8, which is hereby incorporated by reference in its entirety.

H. CBS Activity Assays

The CBS activity was determined by a previously described radioisotope assay using L-[¹⁴C]-serine as the labeled substrate with the following modification. See, Kraus et al., Methods Enzymol 1987, 143, 388-94: Bublil et al., J Clin Invest 2016, 126, (6), 2372-84, each of which are hereby incorporated by reference in their entireties. Four or five μl of undiluted plasma was assayed in a total volume of 100 μl for 30 minutes. The reaction was terminated by mixing of 20 μl of a reaction mixture with 5 μL of chilled performic acid (30% H₂O₂:100% formic acid=1:9) and further processed as described in the original protocol.

I. PEGylation Mapping

Mapping of maleimide and NHS ester PEGylation of htCBS C15S was performed using ESI-QTOF mass spectrometer model 6538 (Agilent, CA, USA). Unmodified enzyme as a control and PEGylated CBS were used as is (unmodified htCBS C15S, 20NHS PEG htCBS-C15S, and 400MA PEG-htCBS C15S) or dePEGylated by incubating the sample at pH 11.5 for 22 hours at 37° C. (20NHS PEG-htCBS C15S). All samples were buffer exchanged, denatured, reduced with 10 mM DTT and alkylated with 30 mM iodoacetamide prior digestion. The 400MA PEG-htCBS C15S and 20NHS PEG-htCBS C15S were digested overnight with Lys-C and Asp-N (1:50 (w/w) ratio, 37° C.), respectively. The 20NHS PEG-htCBS C15S conjugates were also chemically digested using 2-nitro-5-thiocyanobenzoic acid (NTCB) to further differentiate which cysteine residues had been modified. Peptides were separated on a C18 column (Xbridge BEH130 3.5 μm, 4.6×150 mm) using RP-HPLC with UV detection (Agilent), and LC/UV/MS/MS chromatograms were analyzed using MassHunter software (Agilent. CA, USA). Peptide mapping and sequence matching was performed using BioConfirm package (Agilent, CA, USA) with manual validation to ensure correct assignment.

J. Statistical Analysis

All data are presented as mean±standard error of the mean (SEM). Statistical comparisons of 2 groups were conducted using an unpaired, two tailed Students t-test. Statistical analysis of 3 or more factor levels was conducted by ANOVA followed by Tukey's multiple comparison test to determine significance. The p value of less than 0.05 was determined to correspond to statistical significance.

Example 2. Optimizing PEGylation of htCBS C15S

To understand the PEGylation patterns and to optimize the conjugation reaction to yield a more uniform modification, the individual PEG-htCBS C15S species were separated and the residues targeted with the ME-400MA maleimide PEG molecules were later identified (FIG. 2). First, the PEGylation reaction was loaded onto a DEAE Sepharose column equilibrated in a buffer with low conductivity and higher pH (10 mM TRIS.HCl pH 8.5) to enhance binding of all 400MA PEG-htCBS C15S species and ran on a shallow salt gradient (5%, 10-110 mM NaCl) to elute the protein. FIG. 2 shows a chromatogram of such a run and the recovery of two pools, which were subsequently analyzed along with an unmodified htCBS C15S and 400MA PEG-htCBS C15S reaction mixture on SDS-PAGE and native PAGE. Gel analysis of the pools provided evidence that the 400MA PEG-htCBS C15S consisted of at least 3 different PEG-htCBS C15S species.

Pool 1 (P1) was enriched in forms which had a weaker negative charge (FIG. 2) and more PEG moieties attached than those recovered in pool 2 (P2). The predominant 400MA PEG-htCBS C15S conjugate in P1 consisted of a dimeric htCBS C15S, where each protein was modified with ME-400MA to the same extent, most likely 2 PEGs per CBS dimer (homoPEGylated dimer). Moreover, P1 contained a minor 400MA PEG-htCBS C15S species with an additional 1 or 2 PEG moieties. On the other hand, the major 400MA PEG-htCBS C15S form of P2 contained a dimeric enzyme, where just one of the proteins was modified with PEG, i.e. 1 PEG per CBS dimer (hemiPEGylated dimer). Separation of individual 400MA PEG-htCBS C15S species enabled the identification of the sites of maleimide PEGylation using LC-MS.

For phase I, the unmodified htCBS C15S, P1 and P2 were digested with Lys-C. Results of LC-MS are shown in FIG. 3A, FIG. 3B, and FIG. 3C. Out of eight cysteine-containing peptides identified from the reduced/alkylated LC-MS peptide map, seven such peptides yielded similar relative abundances in all three samples indicating that these peptides were unlikely to have been PEGylated. Only one peptide (272-322; SEQ ID NO: 31) containing two cysteine residues (C272 and C275) showed a significant decrease in each of the two 400MA PEG-htCBS C15S species (P1 and P2).

To further differentiate whether C272 and/or C275 were modified in phase II, the two residues would need to be separated into different peptides. Reagent NTCB cleaves N-terminally to a free cysteine residue. Results of LC-MS of an unmodified reference, homoPEGylated, and hemiPEGylated proteins are shown in FIG. 4A. FIG. 4B, and FIG. 4C.

Only one peptide (244-271; SEQ ID NO: 32) showed a significant decrease in 400MA PEG-htCBS C15S forms (more in P1 than for P2) compared to similarly treated unmodified htCBS C15S. The yield for the 244-271 peptide was lowered in the homoPEGylated 400MA PEG-htCBS C15S P1 sample and to a lesser extent in the hemiPEGylated 400MA PEG-htCBS C15S P2.

The peptide recovery signifies that C272 is blocked, thus the lower yield of the 244-271 peptide in 400MA PEG-htCBS C15S forms signifies that C272 is the primary site for maleimide PEGylation. It is possible that both cysteine residues C272 and C275 are involved in the PEGylation of higher molecular weight species present in the low abundance in P1. Low NTCB digestion efficiency resulted in low abundance of the recovered peptides.

Example 3. Reproducibility of PEGylation of htCBS C15S with NHS Ester PEG Molecules

The issues with reproducibility of htCBS C15S PEGylation with maleimide PEGs led to analysis of other PEGylation chemistries, particularly the use of NHS ester-activated PEGs. First, modification of htCBS C15S was assessed with three NHS ester PEG molecules to determine their impact on enzyme activity. Four, three and two different PEG:CBS molar ratios were tested in PEGylations with 5, 10 and 20 kDa linear NHS ester PEG molecules, respectively. Unlike the site-selective maleimide PEGylation, the NHS ester PEG molecules yielded a reproducible mixture of highly modified isomers that were practically inseparable using SDS-PAGE as well as by native PAGE or SEC-HPLC.

For example, htCBS C15S modified by three linear 5, 10, and 20 kDa NHS ester PEG molecules (5NHS, 10NHS, and 20NHS) in different molar excesses of PEG molecules to the CBS protein were separated on SDS-PAGE (BIORAD® 10% Mini-PROTEAN® TGX gel) or native PAGE (BIORAD® 4-15% Mini-PROTEAN® TGX gel) and stained with Safe Stain (Invitrogen) per manufacturer's recommendation. Partial separation was achieved only using NR-CE (FIG. 1), which served as a method for “fingerprinting” of the 20NHS PEG-htCBS C15S production batches.

While a 10-20-fold molar excess of PEG molecules to the CBS protein yielded NHS PEG-htCBS C15S species with only a slight effect on CBS activity, higher excess of PEG, such as 40- and 60-fold for 5 kDa and 40-fold for 10 kDa NHS PEG, resulted in a markedly reduced activity of the respective NHS PEG-htCBS C15S conjugates (FIG. 5). Thus, conjugation of htCBS C15S with either NHS PEG molecules in a PEGylation reaction with no more than 20-fold PEG:CBS molar excess yielded highly modified, bulky NHS PEG-htCBS C15S conjugates with essentially fully retained activity. PEGylation of htCBS C15S with lysine-targeting 5, 10 or 20 kDa NHS ester PEGs used in up to a 20-fold molar excess to htCBS C15S protein did not significantly affect the enzyme's specific activity (FIG. 5), a property shared with the maleimide PEGs (maleimide PEGylation data shown in Bublil et al., J Clin Invest 2016, 126, (6), 2372-84 and International application PCT/2016/061050, which are hereby incorporated by reference in their entireties).

PEGylation of htCBS C15S with 20NHS ester PEG molecules showed reproducibility. Similarly, as done for the maleimide PEGs, the reproducibility of htCBS C15S PEGylation with 20NHS PEG (10-fold PEG:CBS molar excess) was assessed. Initial batches had a highly variable extent of PEGylation (data not shown). However, the main culprit of variability was identified as the residual ammonium sulfate in unmodified htCBS C15S. An extensive buffer exchange scheme was employed at the end of the chromatographic purification (15-20 diavolumes) to formulate the enzyme into the pre-PEGylation buffer, which resulted in a reproducible PEGylating pattern.

Four batches of purified unmodified htCBS C15S were PEGylated with 20NHS PEG molecules yielding four 20NHS PEG-htCBS C15S conjugates. The respective 4 pairs of unmodified enzyme (pre) and 20NHS PEG-htCBS C15S conjugate (post) were designated as LAB, 10L, TR1 and TR2. The protein samples were resolved in SDS-PAGE (BIORAD® 10% Mini-PROTEAN® TGX gel) or native PAGE (BIORAD® 4-15% Mini-PROTEAN® TGX gel) and stained with Safe Stain (Invitrogen) per manufacturer's recommendation or iodine stained to detect PEG. Unmodified htCBS C15S and free unreacted PEG molecules were also observed.

Compared to all the previous PEG-htCBS C15S conjugates, the 20NHS PEG-htCBS C15S showed increased viscosity (data not shown) and thus there was a concern of sufficient free, unreacted PEG molecule removal during the final (post PEGylation) buffer exchange. Indeed, processing of the initial batches (for example LAB and 10L) yielded a large amount of unreacted PEG molecules carried over to the final product. Corrective measures, which included removal of 20% DMSO from the mixture, higher MWCO membrane cartridge for TFF unit (100 versus 30 kDa), 3-fold larger surface area and 2-fold dilution of the PEGylation mixture prior buffer exchange, all helped to reduce the viscosity and assisted in better clearance of free PEG molecules during the final formulation (TR1 and TR2).

Example 4. Characterizing the Extent of PEGylation Using NHS Ester PEG Molecules

SDS and native PAGE were not observed to sufficiently resolve individual 20NHS PEG-htCBS C15S species. Two additional methods were developed for characterization of the extent to which the htCBS C15S was modified with 20NHS PEG molecules allowing for a quick assessment of PEGylation in production and providing a definition of acceptance criteria for the product (FIG. 1).

One method relied on comparison of SEC-HPLC chromatographic profiles of individual 20NHS PEG-htCBS C15S batches compared with that of unmodified htCBS C15S as shown in Table 1.

TABLE 1 UV 225 nm Absorbance [AU] values for different batches Batch identifier Time unmodified [min] htCBS C15S 6-70 6-89 8-14 8-15 8-16 8-22 8-23 8-24 8-25 0.00 2.30E−05 2.00E−06 1.00E−05 1.00E−06 1.30E−05 8.00E−06 −1.20E−05 −3.30E−05 −1.00E−06 1.00E−05 0.02 2.70E−05 4.00E−06 5.00E−06 2.00E−06 4.00E−06 −1.20E−05 1.00E−06 −1.50E−05 −3.20E−05 6.00E−06 0.03 2.10E−05 −1.00E−06 1.80E−05 6.00E−06 1.80E−05 5.00E−06 −1.60E−05 −2.30E−05 −1.30E−05 1.50E−05 0.05 1.40E−05 −6.00E−06 1.50E−05 4.00E−06 5.00E−06 5.00E−06 −6.00E−06 −1.50E−05 −2.40E−05 6.00E−06 0.07 1.20E−05 1.60E−05 3.30E−05 8.00E−06 5.00E−06 8.00E−06 3.00E−06 −2.10E−05 −1.80E−05 6.00E−06 0.08 1.20E−05 1.20E−05 7.00E−06 4.00E−06 −5.00E−06 1.40E−05 6.00E−06 6.00E−06 −7.00E−06 4.00E−06 0.10 1.30E−05 −8.00E−06 1.70E−05 1.20E−05 7.00E−06 −1.00E−06 1.00E−06 −1.50E−05 −2.30E−05 6.00E−06 0.12 3.10E−05 1.30E−05 4.00E−06 1.00E−06 1.50E−05 8.00E−06 2.00E−06 5.00E−06 −1.30E−05 1.00E−05 0.13 2.70E−05 4.00E−06 1.00E−05 2.70E−05 1.40E−05 1.80E−05 1.20E−05 −6.00E−06 −2.50E−05 1.10E−05 0.15 2.20E−05 1.00E−06 2.40E−05 1.30E−05 −1.00E−06 2.30E−05 2.00E−06 0.00E+00 −1.80E−05 7.00E−06 0.17 1.60E−05 1.40E−05 1.20E−05 2.20E−05 1.20E−05 1.00E−06 −3.00E−06 5.00E−06 −1.20E−05 1.80E−05 0.18 1.10E−05 −4.00E−06 1.30E−05 1.00E−05 −1.00E−06 −3.00E−06 3.00E−06 6.00E−06 −1.80E−05 1.50E−05 0.20 1.40E−05 2.00E−06 1.50E−05 3.00E−06 1.60E−05 1.60E−05 −8.00E−06 1.20E−05 −2.30E−05 1.80E−05 0.22 2.10E−05 1.30E−05 1.00E−05 3.40E−05 1.00E−05 7.00E−06 0.00E+00 5.00E−06 −2.70E−05 1.60E−05 0.23 1.70E−05 5.00E−06 8.00E−06 1.00E−06 1.00E−06 1.70E−05 1.10E−05 1.00E−06 −2.30E−05 1.80E−05 0.25 1.70E−05 1.20E−05 2.50E−05 1.00E−05 6.00E−06 5.00E−06 −1.30E−05 −7.00E−06 −3.20E−05 2.50E−05 0.27 8.00E−06 −1.00E−06 2.70E−05 1.20E−05 1.40E−05 1.30E−05 −1.20E−05 −4.00E−06 −2.70E−05 2.40E−05 0.28 2.20E−05 2.00E−06 8.00E−06 2.10E−05 −3.00E−06 2.00E−06 7.00E−06 1.30E−05 −2.80E−05 3.10E−05 0.30 2.60E−05 −3.00E−06 1.80E−05 2.60E−05 5.00E−06 1.40E−05 −6.00E−06 7.00E−06 −3.10E−05 2.00E−05 0.32 1.70E−05 7.00E−06 2.40E−05 −4.00E−06 5.00E−06 1.00E−06 2.00E−06 −7.00E−06 −3.10E−05 1.50E−05 0.33 1.40E−05 5.00E−06 2.20E−05 3.10E−05 1.30E−05 1.00E−05 2.00E−06 8.00E−06 −3.00E−05 1.50E−05 0.35 1.50E−05 6.00E−06 1.10E−05 2.00E−06 6.00E−06 6.00E−06 −5.00E−06 7.00E−06 −1.80E−05 2.30E−05 0.37 8.00E−06 1.00E−06 4.00E−06 1.80E−05 0.00E+00 2.80E−05 −3.00E−06 4.00E−06 −3.80E−05 2.40E−05 0.38 2.00E−06 0.00E+00 4.40E−05 2.70E−05 1.00E−05 1.60E−05 2.00E−06 3.00E−06 −3.40E−05 3.10E−05 0.40 1.00E−05 1.30E−05 4.00E−05 7.00E−06 4.00E−06 1.00E−06 1.10E−05 1.70E−05 −3.50E−05 3.40E−05 0.42 1.40E−05 −3.00E−06 1.80E−05 4.00E−06 5.00E−06 2.00E−05 1.00E−05 0.00E+00 −3.40E−05 3.40E−05 0.43 6.00E−06 −4.00E−06 2.20E−05 1.00E−05 1.60E−05 2.10E−05 5.00E−06 −3.00E−06 −3.80E−05 2.70E−05 0.45 1.00E−05 −1.00E−05 1.80E−05 1.20E−05 3.30E−05 1.10E−05 1.10E−05 −8.00E−06 −2.60E−05 3.30E−05 0.47 6.00E−06 −1.10E−05 2.00E−05 2.00E−06 0.00E+00 1.10E−05 2.40E−05 −1.20E−05 −2.80E−05 2.50E−05 0.48 1.20E−05 1.10E−05 2.00E−05 1.60E−05 2.20E−05 1.80E−05 6.00E−06 −4.00E−06 −3.00E−05 3.70E−05 0.50 6.00E−06 2.00E−06 2.10E−05 1.60E−05 4.00E−06 1.20E−05 3.20E−05 −7.00E−06 −3.30E−05 3.10E−05 0.52 3.20E−05 1.20E−05 1.70E−05 4.00E−06 0.00E+00 6.00E−06 1.00E−05 −1.10E−05 −3.50E−05 2.40E−05 0.53 1.50E−05 7.00E−06 2.50E−05 2.30E−05 4.00E−06 0.00E+00 2.30E−05 0.00E+00 −3.30E−05 2.40E−05 0.55 1.10E−05 7.00E−06 2.80E−05 2.60E−05 4.00E−06 1.60E−05 3.60E−05 −2.00E−05 −4.00E−05 2.70E−05 0.57 8.00E−06 −4.00E−06 5.20E−05 1.00E−05 3.00E−06 8.00E−06 1.50E−05 −2.00E−05 −2.10E−05 5.40E−05 0.58 1.50E−05 1.20E−05 3.00E−05 6.00E−06 1.20E−05 5.00E−06 1.80E−05 −1.60E−05 −2.50E−05 2.30E−05 0.60 1.20E−05 1.00E−05 4.50E−05 5.00E−06 −2.00E−06 2.00E+05 2.30E−05 −1.80E−05 −1.30E−05 2.00E−05 0.62 2.00E−06 2.70E−05 3.50E−05 3.20E−05 7.00E−06 2.00E−06 3.10E−05 −1.80E−05 −3.20E−05 3.10E−05 0.63 1.40E−05 −8.00E−06 4.30E−05 1.10E−05 1.40E−05 1.30E−05 2.80E−05 −2.80E−05 −3.80E−05 3.00E−05 0.65 7.00E−06 −7.00E−06 3.80E−05 1.30E−05 3.00E−06 1.40E−05 2.60E−05 −2.10E−05 −3.10E−05 2.70E−05 0.67 1.00E−06 −1.60E−05 4.20E−05 2.80E−05 −1.00E−06 8.00E−06 2.10E−05 −2.70E−05 −1.70E−05 4.10E−05 0.68 2.30E−05 0.00E+00 3.80E−05 2.20E−05 4.00E−06 1.60E−05 2.40E−05 −2.10E−05 −2.30E−05 3.10E−05 0.70 1.50E−05 −1.00E−06 3.30E−05 2.60E−05 3.00E−06 1.20E−05 2.80E−05 −3.10E−05 −3.40E−05 2.70E−05 0.72 −1.00E−06 −4.00E−06 2.00E−05 1.50E−05 1.10E−05 3.50E−05 1.50E−05 −2.60E−05 −3.20E−05 3.80E−05 0.73 7.00E−06 3.00E−06 4.00E−05 1.60E−05 1.40E−05 1.10E−05 3.20E−05 −2.30E−05 −2.30E−05 4.50E−05 0.75 1.80E−05 4.00E−06 4.50E−05 3.00E−05 1.30E−05 1.40E−05 2.80E−05 −3.30E−05 −1.50E−05 3.20E−05 0.77 1.60E−05 3.00E−06 3.40E−05 1.30E−05 1.00E−05 7.00E−05 4.00E−05 −2.00E−05 −3.80E−05 2.70E−05 0.78 3.10E−05 −1.20E−05 3.60E−05 1.10E−05 5.00E−06 5.00E−06 3.70E−05 −3.20E−05 −2.80E−05 3.40E−05 0.80 5.00E−06 −1.00E−05 3.60E−05 1.80E−05 6.00E−06 1.30E−05 2.40E−05 −2.20E−05 −2.10E−05 3.00E−05 0.82 8.00E−06 −1.00E−05 3.10E−05 2.20E−05 1.00E−05 7.00E−06 2.70E−05 −2.10E−05 −3.10E−05 4.30E−05 0.83 3.80E−05 −1.00E−06 3.70E−05 2.30E−05 3.00E−06 1.10E−05 3.40E−05 −3.00E−05 −3.50E−05 2.40E−05 0.85 1.60E−05 2.00E−06 5.10E−05 2.20E−05 1.50E−05 2.20E−05 2.50E−05 −2.80E−05 −2.80E−05 4.00E−05 0.87 4.00E−06 1.00E−06 3.60E−05 1.40E−05 3.00E−06 1.40E−05 3.40E−05 −3.70E−05 −2.30E−05 4.80E−05 0.88 3.50E−05 7.00E−06 4.60E−05 3.80E−05 1.70E−05 1.20E−05 3.40E−05 −3.00E−05 −1.40E−05 3.70E−05 0.90 3.10E−05 6.00E−06 4.70E−05 2.60E−05 1.70E−05 5.00E−06 3.40E−05 −3.60E−05 −2.10E−05 3.30E−05 0.92 1.80E−05 −1.10E−05 5.10E−05 2.20E−05 1.20E−05 −2.00E−06 3.30E−05 −2.50E−05 −1.40E−05 3.40E−05 0.93 2.20E−05 −7.00E−06 4.20E−05 2.00E−05 2.00E−05 1.60E−05 3.80E−05 −2.50E−05 −1.20E−05 7.50E−05 0.95 1.10E−05 2.00E−06 3.50E−05 2.10E−05 1.40E−05 4.00E−06 3.20E−05 −2.80E−05 −1.20E−05 4.20E−05 0.97 1.70E−05 4.00E−06 3.60E−05 4.60E−05 −3.00E−06 −1.00E−06 3.40E−05 −4.40E−05 −1.50E−05 4.20E−05 0.98 1.50E−05 1.20E−05 5.50E−05 3.30E−05 1.60E−05 2.50E−05 3.80E−05 −3.60E−05 −1.00E−06 4.50E−05 1.00 1.30E−05 7.00E−06 5.10E−05 3.50E−05 1.60E−05 3.60E−05 4.00E−05 −3.80E−05 −2.00E−05 3.80E−05 1.02 6.00E−06 −1.00E−06 3.40E−05 3.00E−05 6.00E−06 7.00E−06 3.40E−05 −5.00E−06 −1.50E−05 2.60E−05 1.03 1.30E−05 2.00E−06 3.70E−05 5.20E−05 1.30E−05 2.00E−05 3.50E−05 −4.30E−05 −3.00E−06 2.10E−05 1.05 1.60E−05 −5.00E−06 4.60E−05 2.60E−05 1.20E−05 1.40E−05 5.40E−05 −3.40E−05 −2.10E−05 4.10E−05 1.07 2.20E−05 −1.00E−06 4.20E−05 3.10E−05 1.30E−05 8.00E−06 4.50E−05 −3.60E−05 −2.40E−05 4.40E−05 1.08 3.20E−05 −1.00E−05 3.50E−05 4.50E−05 1.20E−05 4.00E−06 4.60E−05 −4.30E−05 −1.80E−05 3.70E−05 1.10 2.10E−05 −1.10E−05 2.80E−05 3.00E−05 2.60E−05 8.00E−06 4.10E−05 −4.00E−05 −1.50E−05 2.80E−05 1.12 2.70E−05 2.00E−06 5.40E−05 2.40E−05 3.00E−06 1.10E−05 6.10E−05 −3.20E−05 −2.50E−05 3.60E−05 1.13 8.00E−06 −1.10E−05 4.30E−05 3.60E−05 6.00E−06 1.20E−05 4.10E−05 −3.60E−05 −2.10E−05 2.30E−05 1.15 3.00E−05 −1.20E−05 5.40E−05 2.70E−05 2.50E−05 1.80E−05 4.00E−05 −4.20E−05 −3.50E−05 2.80E−05 1.17 2.60E−05 3.00E−06 6.30E−05 2.20E−05 2.60E−05 1.40E−05 4.20E−05 −3.60E−05 −2.70E−05 3.40E−05 1.18 3.00E−05 8.00E−06 3.70E−05 2.30E−05 1.40E−05 1.50E−05 5.10E−05 −4.50E−05 −3.60E−05 4.20E−05 1.20 2.60E−05 −2.00E−06 4.40E−05 2.10E−05 2.00E−05 2.00E−05 4.10E−05 −4.10E−05 −2.50E−05 3.00E−05 1.22 1.70E−05 1.00E−06 5.50E−05 3.10E−05 6.00E−06 5.00E−06 4.00E−05 −2.30E−05 −2.20E−05 3.40E−05 1.23 1.60E−05 7.00E−06 6.20E−05 2.60E−05 3.10E−05 4.00E−06 6.30E−05 −3.80E−05 −2.40E−05 2.80E−05 1.25 −1.00E−06 −1.10E−05 5.10E−05 3.10E−05 3.00E−06 1.60E−05 5.50E−05 −3.80E−05 −2.50E−05 3.10E−05 1.27 5.00E−06 −1.00E−06 5.00E−05 3.00E−05 1.50E−05 2.20E−05 6.10E−05 −3.40E−05 −1.70E−05 2.60E−05 1.28 1.10E−05 −1.10E−05 4.20E−05 1.20E−05 1.60E−05 1.40E−05 6.40E−05 −3.60E−05 −2.20E−05 3.00E−05 1.30 1.80E−05 −3.00E−06 5.70E−05 2.10E−05 1.40E−05 1.20E−05 5.20E−05 −3.10E−05 −1.10E−05 3.00E−05 1.32 2.70E−05 −1.00E−05 5.00E−05 2.10E−05 2.20E−05 1.50E−05 4.60E−05 −2.00E−05 −3.00E−05 3.40E−05 1.33 2.50E−05 −7.00E−06 4.30E−05 3.10E−05 2.00E−05 1.20E−05 6.50E−05 −3.40E−05 −3.40E−05 2.10E−05 1.35 1.20E−05 −1.00E−06 5.40E−05 2.30E−05 2.80E−05 1.80E−05 6.40E−05 −4.30E−05 −2.00E−05 2.30E−05 1.37 1.60E−05 2.00E−06 5.00E−05 2.30E−05 4.30E−05 0.00E−00 7.50E−05 −4.20E−05 −1.40E−05 2.40E−05 1.38 1.60E−05 −3.00E−06 4.70E−05 1.40E−05 1.00E−05 1.20E−05 7.30E−05 −3.50E−05 −2.00E−05 2.70E−05 1.40 2.80E−05 5.00E−06 5.00E−05 2.40E−05 1.70E−05 3.30E−05 7.00E−05 −3.10E−05 −1.60E−05 4.00E−05 1.42 8.00E−06 3.00E−06 5.20E−05 1.20E−05 2.30E−05 2.00E−05 7.10E−05 −4.50E−05 −2.30E−05 3.50E−05 1.43 2.60E−05 6.00E−06 6.30E−05 3.00E−06 2.40E−05 2.80E−05 6.50E−05 −4.10E−05 −1.80E−05 2.40E−05 1.45 3.70E−05 −4.00E−06 4.60E−05 2.60E−05 2.20E−05 2.30E−05 7.20E−05 −1.80E−05 −3.30E−05 2.60E−05 1.47 1.70E−05 −1.10E−05 5.50E−05 1.70E−05 2.50E−05 1.10E−05 6.30E−05 −3.00E−05 −2.10E−05 2.80E−05 1.48 1.80E−05 2.10E−05 6.00E−05 2.60E−05 2.80E−05 3.70E−05 6.60E−05 −4.30E−05 −1.30E−05 1.20E−05 1.50 3.50E−05 0.00E+00 5.30E−05 2.30E−05 2.70E−05 2.40E−05 5.40E−05 −3.30E−05 −8.00E−06 3.50E−05 1.52 2.80E−05 1.10E−05 6.10E−05 1.70E−05 2.20E−05 4.50E−05 7.30E−05 −3.40E−05 −3.10E−05 3.20E−05 1.53 2.80E−05 1.00E−06 5.70E−05 8.00E−06 2.10E−05 4.30E−05 4.30E−05 −4.50E−05 −1.30E−05 2.40E−05 1.55 1.80E−05 1.00E−05 4.50E−05 1.50E−05 1.80E−05 4.60E−05 5.20E−05 −2.40E−05 −3.50E−05 3.70E−05 1.57 6.00E−06 1.00E−06 5.10E−05 1.70E−05 1.10E−05 5.20E−05 4.40E−05 −2.40E−05 −1.00E−05 1.00E−05 1.58 4.30E−06 2.00E−06 6.60E−05 1.80E−05 1.80E−05 4.40E−05 3.50E−05 −4.10E−05 −1.70E−05 3.20E−05 1.60 3.70E−05 1.00E−06 5.20E−05 5.00E−06 2.30E−05 5.00E−05 4.60E−05 −2.80E−05 −1.70E−05 2.80E−05 1.62 1.70E−05 7.00E−06 5.20E−05 6.00E−06 1.50E−05 5.10E−05 4.10E−05 −3.70E−05 −3.00E−06 1.40E−05 1.63 3.50E−05 1.40E−05 4.50E−05 7.00E−06 4.10E−05 5.30E−05 3.40E−05 −3.80E−05 −1.00E−05 3.50E−05 1.65 2.20E−05 4.00E−06 4.40E−05 1.40E−05 2.60E−05 5.10E−05 4.70E−05 −5.00E−05 −2.10E−05 1.30E−05 1.67 1.20E−05 2.10E−05 5.50E−05 2.40E−05 2.00E−05 4.60E−05 4.10E−05 −3.10E−05 −2.10E−05 2.10E−05 1.68 2.30E−05 1.40E−05 5.20E−05 4.00E−06 4.00E−05 7.60E−05 3.70E−05 −2.80E−05 −6.00E−06 3.10E−05 1.70 3.00E−06 1.80E−05 4.50E−05 1.70E−05 1.60E−05 5.30E−05 3.80E−05 −2.50E−05 −1.80E−05 1.50E−05 1.72 1.70E−05 1.70E−05 5.00E−05 2.10E−05 1.60E−05 6.10E−05 4.00E−05 −3.40E−05 −1.40E−05 1.30E−05 1.73 3.50E−05 8.00E−06 5.60E−05 1.30E−05 3.00E−05 5.00E−05 4.70E−05 −3.60E−05 −2.20E−05 7.00E−06 1.75 2.60E−05 7.00E−06 5.00E−05 2.00E−05 1.30E−05 5.70E−05 4.10E−05 −3.20E−05 −1.00E−05 2.50E−05 1.77 1.70E−05 1.10E−05 5.00E−05 2.40E−05 3.00E−05 5.60E−05 2.10E−05 −3.40E−05 −2.00E−05 7.00E−06 1.78 3.10E−05 8.00E−06 4.60E−05 2.40E−05 3.40E−05 4.20E−05 3.50E−05 −3.00E−05 −3.10E−05 7.00E−06 1.80 4.00E−05 1.10E−05 4.50E−05 3.70E−05 4.00E−05 4.30E−05 3.20E−05 −3.40E−05 −3.10E−05 2.70E−05 1.82 1.80E−05 1.10E−05 4.00E−05 1.70E−05 2.60E−05 6.00E−05 4.00E−05 −2.20E−05 −2.70E−05 3.30E−05 1.83 7.00E−06 1.30E−05 5.30E−05 2.00E−05 1.30E−05 4.80E−05 4.60E−05 −4.10E−05 −5.00E−06 1.80E−05 1.85 2.10E−05 1.40E−05 6.10E−05 1.70E−05 1.70E−05 4.10E−05 5.50E−05 −4.00E−05 −6.00E−06 1.70E−05 1.87 1.80E−05 1.80E−05 4.60E−05 2.70E−05 3.50E−05 3.60E−05 4.00E−05 −2.60E−05 −1.20E−05 2.10E−05 1.88 4.30E−05 2.50E−05 4.30E−05 1.80E−05 2.70E−05 5.00E−05 2.70E−05 −5.30E−05 −2.30E−05 3.10E−05 1.90 5.00E−05 2.20E−05 4.50E−05 2.40E−05 3.20E−05 5.50E−05 4.00E−05 −3.70E−05 −1.80E−05 1.30E−05 1.92 2.20E−05 1.70E−05 6.10E−05 2.40E−05 2.50E−05 4.60E−05 2.50E−05 −2.70E−05 −1.80E−05 2.60E−05 1.93 3.10E−05 2.50E−05 6.70E−05 2.80E−05 2.60E−05 4.10E−05 2.80E−05 −4.00E−05 −2.50E−05 3.00E−05 1.95 2.70E−05 1.70E−05 6.30E−05 3.60E−05 2.50E−05 2.40E−05 3.00E−05 −4.70E−05 −2.80E−05 1.80E−05 1.97 2.40E−05 1.60E−05 4.60E−05 2.50E−05 2.70E−05 3.70E−05 1.80E−05 −4.20E−05 −3.00E−05 1.80E−05 1.98 3.20E−05 1.80E−05 3.30E−05 1.20E−05 1.80E−05 4.50E−05 4.00E−05 −4.60E−05 −1.10E−05 5.00E−05 2.00 3.10E−05 2.30E−05 5.10E−05 1.80E−05 2.30E−05 4.10E−05 3.60E−05 −4.00E−05 −1.20E−05 2.40E−05 2.02 2.70E−05 2.30E−05 4.10E−05 2.20E−05 1.70E−05 5.10E−05 2.40E−05 −3.30E−05 −1.60E−05 3.60E−05 2.03 2.30E−05 2.00E−05 5.00E−05 2.00E−05 2.30E−05 5.10E−05 2.60E−05 −4.30E−05 −6.00E−06 3.20E−05 2.05 1.20E−05 2.20E−05 3.00E−05 2.80E−05 2.30E−05 4.70E−05 3.60E−05 −3.80E−05 −1.70E−05 3.60E−05 2.07 2.70E−05 1.70E−05 5.00E−05 1.40E−05 2.60E−05 4.30E−05 3.80E−05 −3.10E−05 −2.70E−05 3.70E−05 2.08 2.20E−05 2.70E−05 5.00E−05 1.30E−05 2.30E−05 5.50E−05 2.10E−05 −2.30E−05 −1.20E−05 3.30E−05 2.10 3.50E−05 2.30E−05 5.20E−05 1.70E−05 3.30E−05 3.40E−05 3.40E−05 −4.00E−05 −2.50E−05 4.80E−05 2.12 2.20E−05 2.80E−05 3.80E−05 2.10E−05 2.40E−05 4.20E−05 2.50E−05 −3.40E−05 −2.50E−05 3.80E−05 2.13 1.70E−05 1.80E−05 5.30E−05 1.80E−05 2.40E−05 5.70E−05 2.10E−05 −6.00E−05 −2.30E−05 3.50E−05 2.15 2.20E−05 1.20E−05 5.40E−05 1.40E−05 2.80E−05 4.80E−05 2.50E−05 −4.30E−05 −1.60E−05 6.70E−05 2.17 3.40E−05 2.00E−05 4.60E−05 6.00E−06 2.80E−05 2.70E−05 2.00E−05 −4.00E−05 −1.10E−05 4.40E−05 2.18 3.20E−05 2.70E−05 6.10E−05 1.60E−05 2.70E−05 4.80E−05 3.20E−05 −4.80E−05 2.00E−06 4.50E−05 2.20 2.30E−05 2.40E−05 5.30E−05 1.80E−05 2.80E−05 5.00E−05 1.80E−05 −4.50E−05 −1.40E−05 3.10E−05 2.22 1.20E−05 2.10E−05 3.80E−05 7.00E−06 2.60E−05 5.20E−05 2.70E−05 −4.00E−05 −1.80E−05 3.20E−05 2.23 1.80E−05 7.00E−06 3.30E−05 −1.00E−06 4.70E−05 4.40E−05 2.10E−05 −4.00E−05 −2.80E−05 3.60E−05 2.25 2.60E−05 2.80E−05 4.20E−05 −3.00E−06 3.20E−05 4.80E−05 2.40E−05 −4.20E−05 −1.70E−05 4.60E−05 2.27 2.50E−05 1.40E−05 5.00E−05 2.00E−05 3.20E−05 3.80E−05 5.00E−06 −2.60E−05 −1.50E−05 4.10E−05 2.28 2.40E−05 1.30E−05 4.30E−05 8.00E−06 8.00E−06 5.70E−05 2.00E−05 −4.20E−05 −6.00E−06 3.10E−05 2.30 3.20E−05 3.00E−05 4.60E−05 −1.00E−06 3.40E−05 4.40E−05 2.30E−05 −3.80E−05 −2.60E−05 1.60E−05 2.32 2.60E−05 2.70E−05 3.00E−05 1.10E−05 4.10E−05 5.30E−05 1.60E−05 −1.80E−05 −1.60E−05 2.10E−05 2.33 1.60E−05 2.00E−05 4.00E−05 2.00E−05 3.40E−05 4.60E−05 2.20E−05 −3.70E−05 −1.30E−05 2.20E−05 2.35 1.80E−05 2.80E−05 3.80E−05 5.00E−06 3.60E−05 5.00E−05 2.40E−05 −2.60E−05 −2.10E−05 2.30E−05 2.37 2.50E−05 3.30E−05 3.80E−05 1.70E−05 3.70E−05 5.00E−05 2.50E−05 −4.20E−05 −8.00E−06 1.70E−05 2.38 3.20E−05 2.80E−05 4.00E−05 1.30E−05 4.60E−05 6.20E−05 2.50E−05 −5.60E−05 −2.50E−05 2.30E−05 2.40 2.40E−05 2.20E−05 4.50E−05 7.00E−06 4.00E−05 5.50E−05 3.50E−05 −3.30E−05 −2.00E−05 3.00E−05 2.42 1.60E−05 2.40E−05 4.80E−05 −1.60E−05 3.10E−05 5.50E−05 2.50E−05 −3.70E−05 −6.00E−06 2.40E−05 2.43 4.00E−05 2.20E−05 3.30E−05 1.00E−05 3.00E−05 5.80E−05 1.70E−05 −3.20E−05 −1.60E−05 2.00E−05 2.45 3.00E−05 1.30E−05 4.70E−05 1.50E−05 2.40E−05 5.00E−05 3.70E−05 −3.20E−05 −7.00E−06 3.70E−05 2.47 2.80E−05 2.50E−05 3.80E−05 4.00E−06 5.20E−05 6.20E−05 3.40E−05 −3.40E−05 0.00E+00 2.20E−05 2.48 1.70E−05 1.80E−05 4.50E−05 2.00E−06 3.80E−05 6.60E−05 1.80E−05 −4.70E−05 −1.30E−05 2.00E−05 2.50 1.80E−05 7.00E−06 3.30E−05 1.70E−05 3.80E−05 5.20E−05 2.50E−05 −4.80E−05 −8.00E−06 2.40E−05 2.52 2.60E−05 2.60E−05 5.10E−05 2.00E−06 2.50E−05 6.40E−05 2.30E−05 −4.60E−05 −7.00E−06 2.50E−05 2.53 4.50E−05 2.20E−05 3.60E−05 4.00E−06 3.10E−05 4.10E−05 3.00E−05 −3.70E−05 −2.20E−05 1.50E−05 2.55 2.10E−05 1.60E−05 3.80E−05 4.00E−06 5.30E−05 4.80E−05 1.70E−05 −3.40E−05 −5.00E−06 2.20E−05 2.57 3.00E−05 1.80E−05 3.30E−05 −6.00E−06 4.50E−05 4.20E−05 2.60E−05 −4.50E−05 8.00E−06 1.30E−05 2.58 2.80E−05 2.70E−05 3.60E−05 −1.00E−06 4.30E−05 4.40E−05 2.20E−05 −3.10E−05 1.30E−05 2.00E−05 2.60 3.40E−05 1.80E−05 4.70E−05 3.00E−06 4.70E−05 5.30E−05 2.70E−05 −3.30E−05 1.00E−05 2.30E−05 2.62 3.60E−05 2.60E−05 4.40E−05 −1.00E−05 4.80E−05 5.10E−05 2.50E−05 −4.10E−05 1.60E−05 2.20E−05 2.63 2.30E−05 2.10E−05 3.30E−05 1.00E−06 4.50E−05 3.70E−05 1.20E−05 −3.40E−05 −6.00E−06 2.30E−05 2.65 2.00E−05 3.00E−05 2.40E−05 −1.00E−06 4.50E−05 4.30E−05 1.80E−05 −5.20E−05 2.00E−06 1.70E−05 2.67 1.40E−05 3.00E−05 3.30E−05 8.00E−06 3.40E−05 5.80E−05 2.00E−06 −5.70E−05 1.20E−05 1.00E−05 2.68 4.00E−05 1.40E−05 4.40E−05 −1.40E−05 3.00E−05 4.30E−05 1.50E−05 −3.50E−05 4.80E−05 2.30E−05 2.70 2.20E−05 2.60E−05 3.70E−05 −6.00E−06 5.70E−05 4.60E−05 8.00E−06 −4.50E−05 4.00E−06 1.80E−05 2.72 2.50E−05 1.70E−05 3.40E−05 1.00E−06 4.50E−05 3.70E−05 5.00E−06 −4.30E−05 1.40E−05 1.50E−05 2.73 3.10E−05 2.40E−05 5.40E−05 −1.40E−05 3.10E−05 4.50E−05 2.00E−05 −5.00E−05 −4.00E−06 2.50E−05 2.75 1.10E−05 1.80E−05 3.10E−05 −2.00E−06 4.00E−05 4.20E−05 1.20E−05 −2.80E−05 1.50E−05 1.10E−05 2.77 1.70E−05 2.70E−05 4.80E−05 −1.10E−05 3.60E−05 2.80E−05 1.60E−05 −3.40E−05 1.10E−05 2.00E−06 2.78 2.00E−05 4.40E−05 4.80E−05 1.10E−05 4.60E−05 4.70E−05 1.10E−05 −4.70E−05 −3.00E−06 1.80E−05 2.80 3.80E−05 2.00E−05 4.60E−05 −7.00E−06 3.80E−05 3.40E−05 1.50E−05 −3.60E−05 1.80E−05 8.00E−06 2.82 3.50E−05 3.70E−05 3.20E−05 −5.00E−06 4.00E−05 3.60E−05 1.50E−05 −3.40E−05 1.40E−05 1.70E−05 2.83 4.80E−05 1.60E−05 4.00E−05 −8.00E−06 3.40E−05 3.40E−05 1.30E−05 −3.20E−05 1.10E−05 8.00E−06 2.85 1.50E−05 1.30E−05 4.10E−05 −7.00E−06 5.70E−05 2.40E−05 1.60E−05 −3.70E−05 4.00E−06 2.00E−05 2.87 1.50E−05 4.70E−05 3.70E−05 −1.00E−05 3.60E−05 3.50E−05 8.00E−06 −4.10E−05 1.00E−05 2.00E−05 2.88 3.00E−05 2.70E−05 3.60E−05 −1.80E−05 3.10E−05 4.00E−05 1.10E−05 −4.80E−05 −3.00E−06 2.20E−05 2.90 2.60E−05 2.00E−05 3.80E−05 −6.00E−06 5.30E−05 7.70E−05 4.00E−06 −3.00E−05 −1.40E−05 3.20E−05 2.92 1.10E−05 2.40E−05 3.60E−05 −1.60E−05 4.60E−05 2.70E−05 5.00E−06 −4.50E−05 3.00E−06 1.30E−05 2.93 2.70E−05 8.00E−06 3.20E−05 0.00E+00 5.50E−05 3.20E−05 5.00E−06 −3.30E−05 3.00E−06 3.00E−05 2.95 3.50E−05 1.40E−05 3.70E−05 −1.20E−05 4.10E−05 3.80E−05 1.10E−05 −4.40E−05 2.00E−06 1.40E−05 2.97 5.10E−05 1.40E−05 4.10E−05 −1.20E−05 2.80E−05 5.70E−05 5.00E−06 −4.10E−05 −1.10E−05 1.70E−05 2.98 1.70E−05 1.70E−05 3.50E−05 −1.10E−05 5.00E−05 5.60E−05 7.00E−06 −3.80E−05 4.00E−06 7.00E−06 3.00 2.50E−05 1.50E−05 3.60E−05 −1.50E−05 4.50E−05 3.20E−05 1.00E−05 −4.50E−05 −1.80E−05 8.00E−06 3.02 3.30E−05 1.60E−05 4.60E−05 4.00E−06 4.10E−05 4.20E−05 −1.20E−05 −4.50E−05 −1.00E−06 2.20E−05 3.03 1.50E−05 1.30E−05 5.70E−05 −1.10E−05 5.00E−05 3.10E−05 1.00E−05 −5.00E−05 1.00E−06 3.60E−05 3.05 2.20E−05 1.40E−05 2.20E−05 −1.00E−05 5.20E−05 3.20E−05 −1.00E−06 −4.20E−05 −1.20E−05 1.00E−05 3.07 2.60E−05 2.40E−05 4.50E−05 −1.20E−05 4.20E−05 3.20E−05 1.00E−06 −2.70E−05 −1.10E−05 1.80E−05 3.08 1.00E−05 1.20E−05 2.60E−05 −1.00E−06 4.30E−05 3.80E−05 8.00E−06 −4.50E−05 −6.00E−06 2.10E−05 3.10 7.00E−06 1.70E−05 3.20E−05 −5.00E−06 5.70E−05 4.00E−05 1.30E−05 −5.30E−05 −1.20E−05 2.70E−05 3.12 1.00E−05 1.60E−05 3.10E−05 −2.20E−05 4.20E−05 4.50E−05 −4.00E−06 −4.60E−05 −3.00E−06 1.50E−05 3.13 2.40E−05 1.00E−05 3.80E−05 −1.50E−05 4.30E−05 3.20E−05 1.60E−05 −4.30E−05 0.00E+00 2.80E−05 3.15 2.60E−05 2.40E−05 3.40E−05 1.00E−06 4.80E−05 3.10E−05 5.00E−06 −2.50E−05 −1.20E−05 2.20E−05 3.17 8.00E−06 3.20E−05 2.80E−05 0.00E+00 4.80E−05 5.00E−05 1.00E−06 −4.20E−05 −1.30E−05 7.00E−06 3.18 1.80E−05 1.80E−05 3.10E−05 −1.70E−05 4.60E−05 4.60E−05 0.00E+00 −4.10E−05 −8.00E−06 1.50E−05 3.20 1.30E−05 2.20E−05 3.10E−05 −7.00E−06 4.20E−05 4.00E−05 1.40E−05 −4.40E−05 −7.00E−06 2.00E−05 3.22 3.50E−05 2.60E−05 5.30E−05 3.00E−06 5.30E−05 3.40E−05 −1.00E−06 −3.70E−05 −1.40E−05 1.80E−05 3.23 2.40E−05 2.20E−05 4.50E−05 −1.10E−05 4.10E−05 3.70E−05 1.10E−05 −4.00E−05 −1.00E−06 3.00E−05 3.25 1.80E−05 1.10E−05 3.60E−05 −5.00E−06 7.40E−05 3.80E−05 1.80E−05 −2.80E−05 −6.00E−06 2.80E−05 3.27 2.50E−05 1.50E−05 2.60E−05 −5.00E−06 4.80E−05 5.10E−05 6.00E−06 −4.30E−05 −7.00E−06 1.40E−05 3.28 1.40E−05 0.00E+00 5.00E−05 −1.00E−05 4.10E−05 4.30E−05 7.00E−06 −3.80E−05 −2.00E−06 1.80E−05 3.30 3.10E−05 1.10E−05 2.70E−05 −1.40E−05 3.10E−05 5.20E−05 1.20E−05 −4.40E−05 −8.00E−06 3.20E−05 3.32 2.30E−05 7.00E−06 3.20E−05 −1.00E−06 3.50E−05 3.70E−05 1.20E−05 −5.10E−05 −1.00E−06 3.60E−05 3.33 2.60E−05 4.00E−06 2.70E−05 −2.00E−06 3.60E−05 5.00E−05 −2.00E−06 −5.20E−05 −4.00E−06 3.10E−05 3.35 2.60E−05 1.80E−05 3.50E−05 −1.00E−05 4.30E−05 5.50E−05 2.00E−06 −5.80E−05 7.00E−06 2.10E−05 3.37 8.00E−06 2.70E−05 5.00E−05 −1.50E−05 3.60E−05 4.20E−05 1.10E−05 −5.50E−05 1.80E−05 1.80E−05 3.38 2.80E−05 1.80E−05 3.40E−05 −1.80E−05 2.80E−05 3.20E−05 −1.30E−05 −4.00E−05 −1.50E−05 2.80E−05 3.40 2.40E−05 1.30E−05 3.40E−05 0.00E+00 2.50E−05 3.50E−05 1.80E−05 −3.70E−05 −3.00E−06 2.00E−05 3.42 1.70E−05 3.10E−05 4.10E−05 −4.00E−06 3.60E−05 4.10E−05 2.00E−06 −4.00E−05 0.00E+00 3.00E−05 3.43 1.60E−05 5.00E−06 3.70E−05 −1.00E−06 5.10E−05 5.60E−05 1.20E−05 −1.80E−05 8.00E−06 2.70E−05 3.45 2.60E−05 2.00E−05 4.00E−05 −3.00E−06 3.20E−05 3.50E−05 7.00E−06 −4.30E−05 8.00E−06 2.40E−05 3.47 2.30E−05 7.00E−06 3.20E−05 1.00E−06 3.20E−05 2.60E−05 −2.00E−06 −5.00E−05 0.00E+00 4.60E−05 3.48 1.80E−05 3.00E−06 3.50E−05 6.00E−06 5.80E−05 3.60E−05 5.00E−06 −5.50E−05 7.00E−06 3.20E−05 3.50 2.20E−05 1.10E−05 3.50E−05 −4.00E−06 4.60E−05 4.40E−05 1.10E−05 −5.70E−05 1.00E−05 2.50E−05 3.52 2.00E−05 2.10E−05 3.60E−05 1.50E−05 5.10E−05 3.40E−05 5.00E−06 −3.20E−05 7.00E−06 3.30E−05 3.53 1.50E−05 3.20E−05 4.30E−05 1.00E−05 2.80E−05 5.10E−05 1.00E−06 −4.80E−05 −8.00E−06 3.30E−05 3.55 2.00E−05 1.80E−05 3.10E−05 1.80E−05 3.70E−05 3.30E−05 −5.00E−06 −5.80E−05 1.00E−06 3.30E−05 3.57 2.20E−05 1.20E−05 4.60E−05 8.00E−06 2.50E−05 4.50E−05 1.50E−05 −3.50E−05 1.00E−05 1.80E−05 3.58 1.50E−05 8.00E−06 3.30E−05 2.70E−05 3.30E−05 4.40E−05 8.00E−06 −4.40E−05 4.00E−06 3.60E−05 3.60 2.10E−05 6.00E−06 4.50E−05 2.60E−05 4.50E−05 3.40E−05 6.00E−06 −4.20E−05 3.00E−06 3.60E−05 3.62 1.20E−05 1.30E−05 4.40E−05 3.00E−05 4.40E−05 4.20E−05 6.00E−06 −4.30E−05 −1.30E−05 2.10E−05 3.63 1.70E−05 2.50E−05 3.60E−05 3.50E−05 3.40E−05 4.80E−05 −1.00E−05 −4.30E−05 −3.00E−06 2.60E−05 3.65 1.80E−05 2.20E−05 4.20E−05 6.50E−05 3.00E−05 4.30E−05 1.50E−05 −4.80E−05 2.00E−06 3.30E−05 3.67 2.20E−05 1.80E−05 3.60E−05 4.70E−05 3.70E−05 3.00E−05 1.10E−05 −4.30E−05 0.00E+00 3.20E−05 3.68 6.00E−06 2.00E−06 2.60E−05 4.60E−05 3.30E−05 3.30E−05 3.00E−06 −4.40E−05 1.30E−05 4.20E−05 3.70 2.00E−06 2.20E−05 3.10E−05 6.10E−05 5.00E−05 3.30E−05 1.00E−05 −4.10E−05 −5.00E−06 1.30E−05 3.72 1.80E−05 1.60E−05 2.80E−05 6.40E−05 3.00E−05 4.70E−05 8.00E−06 −5.10E−05 2.00E−06 3.60E−05 3.73 2.30E−05 2.40E−05 2.70E−05 6.90E−05 3.30E−05 4.80E−05 5.00E−06 −4.40E−05 8.00E−06 2.50E−05 3.75 2.20E−05 1.30E−05 4.10E−05 7.20E−05 3.10E−05 4.10E−05 1.00E−05 0.00E+00 3.40E−05 3.40E−05 3.77 2.40E−05 2.00E−06 4.10E−05 7.00E−05 4.30E−05 4.50E−05 1.00E−05 −5.20E−05 4.00E−06 4.10E−05 3.78 −1.00E−06 2.30E−05 3.10E−05 1.10E−04 2.40E−05 4.40E−05 1.70E−05 −5.80E−05 3.20E−05 4.50E−05 3.80 2.60E−05 1.10E−05 3.40E−05 7.20E−05 4.00E−05 4.10E−05 1.30E−05 −4.80E−05 1.00E−05 4.30E−05 3.82 2.40E−05 2.80E−05 3.00E−05 6.20E−05 3.40E−05 4.80E−05 1.00E−05 −6.10E−05 2.00E−06 2.40E−05 3.83 1.40E−05 1.80E−05 3.30E−05 5.20E−05 2.30E−05 4.00E−05 1.80E−05 −2.80E−05 6.00E−06 3.60E−05 3.85 2.00E−05 8.00E−06 3.20E−05 6.20E−05 3.40E−05 5.00E−05 1.40E−05 −4.30E−05 2.00E−05 4.30E−05 3.87 1.30E−05 5.00E−06 3.80E−05 4.40E−05 1.70E−05 4.60E−05 5.00E−06 −5.00E−05 2.20E−05 4.00E−05 3.88 1.40E−05 7.00E−06 4.20E−05 4.50E−05 2.60E−05 4.70E−05 2.10E−05 −5.50E−05 1.30E−05 2.80E−05 3.90 2.30E−05 1.40E−05 4.10E−05 3.70E−05 2.50E−05 6.00E−05 2.00E−06 −4.80E−05 1.20E−05 4.00E−05 3.92 1.40E−05 1.60E−05 3.80E−05 4.60E−05 3.00E−05 6.70E−05 2.80E−05 −6.00E−05 1.20E−05 4.40E−05 3.93 1.40E−05 1.40E−05 3.80E−05 3.40E−05 1.70E−05 7.40E−05 1.70E−05 −4.30E−05 2.30E−05 3.20E−05 3.95 1.80E−05 2.60E−05 3.50E−05 3.70E−05 3.40E−05 7.20E−05 3.10E−05 −5.60E−05 1.80E−05 3.10E−05 3.97 2.30E−05 2.30E−05 4.20E−05 3.30E−05 3.80E−05 8.00E−05 2.80E−05 −5.70E−05 1.20E−05 4.40E−05 3.98 1.30E−05 1.70E−05 3.70E−05 4.20E−05 3.30E−05 9.00E−05 3.30E−05 −6.30E−05 1.70E−05 3.50E−05 4.00 4.10E−05 1.20E−05 4.30E−05 2.00E−05 2.30E−05 1.01E−04 4.50E−05 −4.70E−05 3.50E−05 3.60E−05 4.02 8.00E−06 6.00E−06 3.30E−05 3.00E−05 4.00E−05 1.06E−04 5.40E−05 −5.20E−05 1.10E−05 3.50E−05 4.03 2.20E−05 8.00E−06 2.40E−05 1.00E−05 2.30E−05 1.09E−04 6.20E−05 −6.50E−05 2.20E−05 5.00E−05 4.05 1.70E−05 3.00E−06 4.00E−05 2.10E−05 3.30E−05 1.07E−04 7.90E−05 −3.00E−05 1.00E−05 3.20E−05 4.07 2.40E−05 1.00E−05 4.10E−05 1.70E−05 3.10E−05 1.11E−04 8.00E−05 −5.30E−05 1.50E−05 2.40E−05 4.08 2.40E−05 1.00E−05 3.60E−05 1.20E−05 1.80E−05 1.19E−04 9.00E−05 −5.30E−05 2.20E−05 3.70E−05 4.10 2.50E−05 2.30E−05 1.70E−05 2.10E−05 1.60E−05 1.11E−04 1.16E−04 −3.40E−05 1.70E−05 3.80E−05 4.12 1.70E−05 2.30E−05 4.40E−05 8.00E−06 3.30E−05 1.21E−04 9.90E−05 −5.40E−05 1.00E−05 4.00E−05 4.13 1.80E−05 1.40E−05 2.80E−05 2.50E−05 2.20E−05 1.05E−04 1.06E−04 −6.70E−05 1.80E−05 4.00E−05 4.15 2.30E−05 1.40E−05 3.80E−05 2.40E−05 2.10E−05 1.17E−04 1.07E−04 −3.60E−05 2.00E−06 4.80E−05 4.17 5.00E−06 7.00E−06 3.10E−05 1.30E−05 1.80E−05 1.11E−04 1.19E−04 −4.20E−05 1.10E−05 5.20E−05 4.18 2.50E−05 2.40E−05 3.20E−05 −5.00E−06 1.40E−05 1.10E−04 1.16E−04 −4.50E−05 8.00E−06 4.50E−05 4.20 2.30E−05 4.00E−06 2.10E−05 1.10E−05 3.20E−05 1.16E−04 1.26E−04 −5.00E−05 8.00E−06 5.00E−05 4.22 2.60E−05 8.00E−06 4.70E−05 2.00E−06 2.80E−05 9.40E−05 1.36E−04 −5.40E−05 2.10E−05 4.80E−05 4.23 2.30E−05 4.00E−06 2.70E−05 5.00E−06 3.00E−05 1.01E−04 1.33E−04 −5.50E−05 1.30E−05 4.60E−05 4.25 2.80E−05 7.00E−06 3.60E−05 −6.00E−06 2.50E−05 9.40E−05 1.32E−04 −5.80E−05 2.40E−05 5.20E−05 4.27 1.00E−05 5.00E−06 3.30E−05 7.00E−06 2.20E−05 7.40E−05 1.26E−04 −5.00E−05 1.50E−05 4.10E−05 4.28 1.60E−05 5.00E−06 3.00E−05 2.00E−06 2.10E−05 8.50E−05 1.39E−04 −6.50E−05 2.20E−05 3.70E−05 4.30 2.10E−05 3.00E−06 3.70E−05 2.00E−06 1.80E−05 6.70E−05 9.70E−05 −5.20E−05 1.60E−05 2.50E−05 4.32 2.30E−05 1.10E−05 3.70E−05 −8.00E−06 2.60E−05 8.20E−05 9.70E−05 −4.70E−05 1.80E−05 5.80E−05 4.33 3.30E−05 −2.00E−06 4.70E−05 −3.00E−06 2.60E−05 6.60E−05 8.60E−05 −5.50E−05 1.10E−05 4.20E−05 4.35 2.40E−05 7.00E−06 5.00E−05 −7.00E−06 2.20E−05 6.10E−05 9.40E−05 −6.30E−05 4.00E−06 4.60E−05 4.37 1.50E−05 1.40E−05 3.10E−05 0.00E+00 3.20E−05 5.60E−05 8.20E−05 −5.70E−05 1.30E−05 4.40E−05 4.38 2.80E−05 5.00E−06 4.70E−05 1.40E−05 1.10E−05 5.40E−05 7.70E−05 −5.60E−05 8.00E−06 2.70E−05 4.40 1.60E−05 1.00E−05 5.20E−05 −5.00E−06 3.70E−05 4.70E−05 7.50E−05 −4.70E−05 1.10E−05 5.00E−05 4.42 2.70E−05 1.20E−05 4.40E−05 −6.00E−06 1.70E−05 6.00E−05 7.40E−05 −4.80E−05 1.60E−05 4.10E−05 4.43 3.10E−05 2.40E−05 4.60E−05 0.00E+00 1.70E−05 3.60E−05 6.20E−05 −6.20E−05 7.00E−06 3.50E−05 4.45 2.40E−05 1.30E−05 4.70E−05 7.00E−06 1.00E−05 3.70E−05 6.20E−05 −6.10E−05 −1.00E−06 3.10E−05 4.47 4.40E−05 5.00E−06 4.80E−05 −3.00E−06 1.30E−05 5.10E−05 5.50E−05 −4.10E−05 3.70E−05 3.40E−05 4.48 2.10E−05 −1.00E−06 4.50E−05 −1.20E−05 1.60E−05 4.50E−05 5.60E−05 −5.10E−05 3.00E−06 4.50E−05 4.50 2.50E−05 1.20E−05 4.30E−05 −7.00E−06 1.20E−05 4.50E−05 6.00E−05 −5.00E−05 1.30E−05 5.40E−05 4.52 2.40E−05 2.30E−05 5.60E−05 −1.00E−05 1.70E−05 3.20E−05 5.30E−05 −5.80E−05 2.20E−05 5.00E−05 4.53 3.60E−05 6.00E−06 5.80E−05 −1.00E−05 1.20E−05 4.60E−05 5.10E−05 −5.80E−05 8.00E−06 5.10E−05 4.55 3.20E−05 2.50E−05 4.10E−05 3.00E−06 1.60E−05 2.80E−05 4.50E−05 −5.50E−05 2.00E−05 4.80E−05 4.57 2.70E−05 4.00E−06 5.50E−05 −1.00E−05 4.00E−06 1.70E−05 5.10E−05 −5.00E−05 2.00E−06 6.00E−05 4.58 3.80E−05 0.00E+00 4.80E−05 1.00E−06 1.50E−05 7.20E−05 4.10E−05 −3.80E−05 1.00E−05 3.70E−05 4.60 1.80E−05 −3.00E−06 5.50E−05 −5.00E−06 6.00E−06 3.10E−05 5.40E−05 −5.30E−05 4.00E−06 4.10E−05 4.62 1.40E−05 3.00E−06 5.10E−05 −1.00E−06 5.00E−06 1.80E−05 4.40E−05 −6.10E−05 1.30E−05 4.70E−05 4.63 1.20E−05 1.30E−05 5.10E−05 −1.10E−05 6.00E−06 3.00E−05 4.30E−05 −6.00E−05 1.00E−06 5.60E−05 4.65 2.40E−05 2.40E−05 4.30E−05 −7.00E−06 2.10E−05 2.40E−05 2.80E−05 −6.70E−05 1.10E−05 5.30E−05 4.67 2.20E−05 2.30E−05 5.40E−05 −1.60E−05 3.10E−05 2.20E−05 4.70E−05 −6.00E−05 1.00E−06 4.20E−05 4.68 1.60E−05 8.00E−06 5.40E−05 −1.20E−05 1.50E−05 2.20E−05 4.60E−05 −6.90E−05 1.50E−05 3.80E−05 4.70 2.60E−05 1.70E−05 4.30E−05 −2.00E−06 5.00E−06 5.00E−06 4.00E−05 −5.50E−05 5.00E−06 4.70E−05 4.72 2.00E−05 −5.00E−06 5.80E−05 4.00E−06 −1.00E−06 2.10E−05 2.40E−05 −5.30E−05 1.10E−05 4.70E−05 4.73 2.50E−05 1.80E−05 6.10E−05 −1.60E−05 1.30E−05 2.00E−05 4.00E−05 −4.10E−05 1.60E−05 4.10E−05 4.75 2.00E−05 −2.00E−06 5.70E−05 −1.10E−05 4.00E−06 8.00E−06 2.30E−05 −5.00E−05 1.10E−05 3.70E−05 4.77 2.30E−05 0.00E+00 5.70E−05 2.00E−06 1.80E−05 −3.00E−06 3.30E−05 −5.40E−05 7.00E−06 6.00E−05 4.78 1.50E−05 1.50E−05 5.20E−05 −2.00E−06 1.20E−05 7.00E−06 2.80E−05 −5.40E−05 4.00E−06 4.60E−05 4.80 2.10E−05 2.10E−05 4.70E−05 0.00E+00 1.30E−05 2.20E−05 3.10E−05 −3.50E−05 1.20E−05 4.10E−05 4.82 2.50E−05 3.00E−05 6.40E−05 1.00E−06 7.00E−06 1.60E−05 1.50E−05 −5.00E−05 1.60E−05 4.10E−05 4.83 7.00E−06 2.00E−05 6.50E−05 −5.00E−06 2.30E−05 1.00E−05 2.60E−05 −5.60E−05 2.00E−05 4.30E−05 4.85 2.10E−05 1.00E−05 5.60E−05 −3.00E−06 1.10E−05 8.00E−06 3.50E−05 −6.10E−05 5.00E−06 4.40E−05 4.87 2.70E−05 8.00E−06 5.50E−05 3.00E−06 −4.00E−06 1.10E−05 2.40E−05 −5.70E−05 8.00E−06 4.80E−05 4.88 1.70E−05 2.00E−06 5.40E−05 −3.00E−06 0.00E+00 1.40E−05 2.00E−05 −5.00E−05 −2.00E−06 5.10E−05 4.90 1.20E−05 7.00E−06 5.10E−05 3.00E−06 1.40E−05 −4.00E−06 1.80E−05 −6.20E−05 1.60E−05 3.80E−05 4.92 3.50E−05 1.30E−05 5.20E−05 1.00E−06 1.10E−05 1.10E−05 3.70E−05 −5.30E−05 6.00E−06 5.60E−05 4.93 3.20E−05 1.60E−05 6.00E−05 −1.20E−05 3.00E−06 −2.00E−06 1.30E−05 −6.30E−05 −1.00E−06 5.10E−05 4.95 2.30E−05 8.00E−06 6.30E−05 0.00E+00 1.50E−05 −1.00E−06 1.30E−05 −2.60E−05 1.20E−05 4.10E−05 4.97 1.50E−05 7.00E−06 7.00E−05 1.00E−05 8.00E−06 8.00E−06 2.20E−05 −5.60E−05 1.00E−05 4.80E−05 4.98 1.70E−05 3.00E−06 4.60E−05 1.00E−05 1.80E−05 −1.00E−06 1.80E−05 −7.30E−05 3.00E−06 5.70E−05 5.00 2.20E−05 1.70E−05 5.20E−05 −1.00E−06 0.00E+00 1.20E−05 2.20E−05 −5.70E−05 1.40E−05 5.50E−05 5.02 1.80E−05 1.20E−05 6.00E−05 1.30E−05 7.00E−06 3.00E−06 2.20E−05 −5.30E−05 −2.00E−06 5.20E−05 5.03 2.00E−05 1.50E−05 5.10E−05 2.00E−06 1.30E−05 2.00E−06 1.70E−05 −4.60E−05 7.00E−06 4.70E−05 5.05 1.70E−05 1.00E−05 5.40E−05 1.00E−05 1.30E−05 5.00E−06 2.60E−05 −5.80E−05 1.10E−05 6.30E−05 5.07 3.30E−05 2.00E−06 6.10E−05 −2.00E−06 3.00E−06 7.00E−06 1.60E−05 −5.30E−05 1.80E−05 6.00E−05 5.08 2.70E−05 2.10E−05 5.00E−05 1.20E−05 1.00E−06 −1.60E−05 1.50E−05 −6.20E−05 −3.00E−06 5.50E−05 5.10 1.70E−05 2.20E−05 4.50E−05 3.00E−06 1.20E−05 −1.00E−06 4.00E−05 −6.00E−05 5.00E−06 6.50E−05 5.12 1.50E−05 6.00E−06 6.40E−05 0.00E+00 1.00E−05 −1.40E−05 1.70E−05 −4.80E−05 −2.00E−06 5.70E−05 5.13 2.30E−05 7.00E−06 5.80E−05 1.10E−05 7.00E−06 4.00E−06 1.60E−05 −8.50E−05 8.00E−06 4.80E−05 5.15 2.10E−05 1.10E−05 7.00E−05 4.00E−06 1.80E−05 −1.00E−06 1.50E−05 −5.40E−05 6.00E−06 5.40E−05 5.17 2.00E−05 1.40E−05 5.30E−05 2.60E−05 1.60E−05 0.00E+00 2.40E−05 −7.00E−05 −1.40E−05 5.50E−05 5.18 1.60E−05 6.00E−06 7.10E−05 5.00E−06 5.00E−06 0.00E+00 1.70E−05 −6.50E−05 3.00E−06 5.80E−05 5.20 3.00E−06 6.00E−06 6.40E−05 5.00E−06 2.00E−06 4.00E−06 1.40E−05 −6.10E−05 1.40E−05 5.70E−05 5.22 2.50E−05 2.80E−05 5.50E−05 2.40E−05 1.30E−05 3.00E−06 1.40E−05 −5.30E−05 0.00E+00 4.60E−05 5.23 1.20E−05 2.70E−05 6.70E−05 8.00E−06 1.30E−05 1.30E−05 1.60E−05 −5.10E−05 1.00E−05 6.20E−05 5.25 2.80E−05 1.80E−05 6.00E−05 3.00E−06 −3.00E−06 −5.00E−06 1.80E−05 −6.90E−05 1.10E−05 5.80E−05 5.27 1.20E−05 1.10E−05 6.50E−05 2.60E−05 5.00E−06 5.00E−06 2.80E−05 −6.50E−05 1.30E−05 5.30E−05 5.28 1.30E−05 4.00E−06 7.60E−05 1.10E−05 1.40E−05 1.10E−05 1.60E−05 −5.00E−05 4.00E−06 6.10E−05 5.30 2.10E−05 8.00E−06 5.20E−05 1.20E−05 −1.00E−06 −5.00E−06 1.20E−05 −5.80E−05 8.00E−06 4.20E−05 5.32 2.60E−05 1.00E−05 6.30E−05 2.80E−05 1.50E−05 4.00E−06 2.30E−05 −5.30E−05 0.00E+00 5.40E−05 5.33 1.80E−05 2.10E−05 6.50E−05 2.00E−05 1.10E−05 −3.00E−06 1.40E−05 −5.30E−05 1.10E−05 4.50E−05 5.35 1.70E−05 1.20E−05 6.70E−05 1.20E−05 −8.00E−06 7.00E−06 1.40E−05 −5.60E−05 −8.00E−06 6.50E−05 5.37 1.80E−05 3.00E−06 6.40E−05 3.10E−05 7.00E−06 −4.00E−06 2.10E−05 −7.70E−05 −2.00E−06 4.40E−05 5.38 1.60E−05 2.00E−05 6.50E−05 1.40E−05 2.40E−05 −2.00E−06 2.50E−05 −7.00E−05 −4.00E−06 6.60E−05 5.40 1.30E−05 1.20E−05 5.10E−05 5.00E−06 7.00E−06 −2.00E−06 1.50E−05 −6.60E−05 6.00E−06 4.50E−05 5.42 3.80E−05 2.40E−05 7.60E−05 1.10E−05 −1.00E−06 −7.00E−06 6.00E−06 −5.80E−05 7.00E−06 5.10E−05 5.43 6.00E−06 7.00E−06 7.70E−05 2.00E−05 8.00E−06 2.00E−06 2.10E−05 −6.20E−05 −5.00E−06 4.50E−05 5.45 1.30E−05 1.20E−05 7.00E−05 1.30E−05 1.20E−05 −1.00E−06 2.60E−05 −4.70E−05 1.40E−05 6.00E−05 5.47 1.80E−05 1.30E−05 7.50E−05 8.00E−06 1.30E−05 2.00E−06 2.40E−05 −5.40E−05 7.00E−06 5.50E−05 5.48 1.20E−05 2.20E−05 6.30E−05 1.30E−05 7.00E−06 −7.00E−06 2.00E−05 −6.00E−05 8.00E−06 5.00E−05 5.50 1.60E−05 3.20E−05 7.40E−05 2.60E−05 8.00E−06 4.00E−06 2.00E−05 −5.20E−05 1.40E−05 6.30E−05 5.52 1.80E−05 2.40E−05 4.80E−05 8.00E−06 5.00E−06 −4.00E−06 1.80E−05 −5.80E−05 1.40E−05 4.70E−05 5.53 2.30E−05 2.00E−05 6.00E−05 3.00E−06 5.00E−06 7.00E−06 1.80E−05 −3.60E−05 1.00E−06 5.00E−05 5.55 2.40E−05 1.30E−05 7.50E−05 2.20E−05 7.00E−06 3.00E−06 2.20E−05 −5.40E−05 1.00E−06 6.00E−05 5.57 2.80E−05 1.60E−05 6.90E−05 1.50E−05 1.20E−05 1.80E−05 1.30E−05 −5.40E−05 4.00E−06 4.40E−05 5.58 3.00E−06 2.40E−05 7.10E−05 2.50E−05 2.00E−06 −8.00E−06 2.10E−05 −6.50E−05 1.00E−05 6.50E−05 5.60 2.00E−05 2.30E−05 5.40E−05 2.10E−05 −1.00E−05 5.00E−06 1.70E−05 −5.40E−05 0.00E+00 5.50E−05 5.62 1.70E−05 2.00E−05 6.90E−05 1.60E−05 5.00E−06 1.00E−06 2.20E−05 −5.10E−05 6.00E−06 5.80E−05 5.63 2.00E−05 1.30E−05 5.50E−05 3.00E−05 −6.00E−06 −1.10E−05 3.50E−05 −3.40E−05 −4.00E−06 5.30E−05 5.65 2.00E−05 1.40E−05 7.70E−05 1.10E−05 0.00E+00 −4.00E−06 1.30E−05 −4.20E−05 2.00E−06 4.50E−05 5.67 4.00E−06 3.10E−05 7.20E−05 2.50E−05 −1.00E−06 −4.00E−06 2.80E−05 −5.20E−05 8.00E−06 4.60E−05 5.68 1.30E−05 2.10E−05 6.10E−05 8.00E−06 2.00E−06 0.00E+00 2.40E−05 −3.60E−05 5.00E−06 5.50E−05 5.70 1.10E−05 2.50E−05 7.30E−05 1.70E−05 6.00E−06 −6.00E−06 1.80E−05 −4.30E−05 8.00E−06 4.20E−05 5.72 3.50E−05 5.00E−06 6.40E−05 1.70E−05 5.00E−06 −1.00E−06 2.00E−05 −4.10E−05 −2.00E−06 5.00E−05 5.73 1.30E−05 2.20E−05 6.20E−05 2.10E−05 8.00E−06 −1.00E−05 2.30E−05 −5.00E−05 4.00E−06 7.30E−05 5.75 2.20E−05 1.70E−05 7.20E−05 2.60E−05 1.00E−06 2.00E−05 1.80E−05 −5.10E−05 −1.20E−05 4.00E−05 5.77 6.00E−06 2.80E−05 6.20E−05 1.00E−05 1.00E−06 −2.00E−06 2.40E−05 −4.40E−05 −1.00E−06 4.10E−05 5.78 2.00E−05 2.50E−05 4.80E−05 2.20E−05 8.00E−06 2.00E−06 2.20E−05 −3.10E−05 0.00E+00 4.50E−05 5.80 2.00E−05 1.80E−05 6.50E−05 1.30E−05 8.00E−06 1.00E−06 1.10E−05 −5.10E−05 −8.00E−06 3.80E−05 5.82 1.10E−05 1.80E−05 5.50E−05 8.00E−06 4.00E−06 0.00E+00 1.40E−05 −4.10E−05 2.00E−06 4.00E−05 5.83 2.00E−05 1.70E−05 5.30E−05 1.70E−05 1.70E−05 3.00E−06 2.20E−05 −4.30E−05 −4.00E−06 3.60E−05 5.85 1.80E−05 2.50E−05 7.30E−05 2.00E−05 2.30E−05 −1.00E−06 2.00E−05 −3.60E−05 −8.00E−06 5.00E−05 5.87 6.00E−06 2.20E−05 7.30E−05 1.80E−05 −1.00E−06 1.10E−05 8.00E−06 −3.00E−05 4.00E−06 2.80E−05 5.88 1.00E−05 2.70E−05 5.50E−05 1.80E−05 1.40E−05 1.50E−05 1.20E−05 −3.30E−05 0.00E+00 4.10E−05 5.90 1.00E−06 4.30E−05 6.40E−05 6.00E−06 1.00E−06 7.00E−06 7.00E−06 −4.50E−05 2.00E−06 4.20E−05 5.92 2.10E−05 2.70E−05 6.30E−05 2.70E−05 7.00E−06 −1.00E−06 2.40E−05 −3.80E−05 −1.00E−06 5.70E−05 5.93 2.00E−06 3.80E−05 7.50E−05 1.80E−05 1.40E−05 5.00E−06 7.00E−06 −3.10E−05 −5.00E−06 3.30E−05 5.95 3.10E−05 3.40E−05 8.00E−05 1.50E−05 1.70E−05 1.00E−06 1.30E−05 −2.40E−05 −6.00E−06 4.00E−05 5.97 1.00E−05 3.20E−05 6.90E−05 6.00E−06 1.20E−05 6.00E−06 1.20E−05 −3.50E−05 1.00E−06 4.50E−05 5.98 3.30E−05 4.70E−05 6.30E−05 3.10E−05 0.00E+00 1.00E−06 1.50E−05 −4.20E−05 8.00E−06 4.40E−05 6.00 1.30E−05 4.40E−05 7.00E−05 2.00E−05 4.00E−06 4.00E−06 −1.00E−06 −2.70E−05 −1.60E−05 4.30E−05 6.02 2.30E−05 3.50E−05 6.50E−05 2.50E−05 1.60E−05 −7.00E−06 1.20E−05 −2.30E−05 1.50E−05 3.10E−05 6.03 1.50E−05 5.00E−05 6.50E−05 1.20E−05 1.00E−05 1.10E−05 6.00E−06 −1.40E−05 −1.00E−06 4.10E−05 6.05 1.80E−05 3.00E−05 7.00E−05 2.00E−05 7.00E−06 7.00E−06 −6.00E−06 −2.10E−05 2.00E−05 2.80E−05 6.07 2.10E−05 3.50E−05 7.60E−05 1.70E−05 4.00E−06 1.30E−05 6.00E−06 −2.70E−05 −4.00E−06 4.60E−05 6.08 1.50E−05 3.20E−05 7.70E−05 2.50E−05 −4.00E−06 4.00E−06 1.30E−05 −3.50E−05 −1.40E−05 5.20E−05 6.10 1.60E−05 2.50E−05 7.20E−05 3.60E−05 1.80E−05 7.00E−06 1.10E−05 −2.60E−05 −1.60E−05 4.50E−05 6.12 1.70E−05 5.70E−05 7.60E−05 6.00E−06 1.00E−06 6.00E−06 6.00E−06 −2.20E−05 −1.80E−05 4.40E−05 6.13 1.20E−05 3.40E−05 6.90E−05 1.70E−05 −1.10E−05 3.00E−06 4.00E−06 −2.50E−05 −1.20E−05 6.00E−05 6.15 1.60E−05 3.60E−05 6.70E−05 1.50E−05 −1.00E−06 −2.00E−06 6.00E−06 −2.60E−05 −2.00E−06 3.50E−05 6.17 4.20E−05 3.10E−05 7.10E−05 1.20E−05 1.40E−05 3.00E−06 1.10E−05 −1.80E−05 −1.00E−05 4.80E−05 6.18 2.00E−05 3.00E−05 8.70E−05 1.40E−05 1.20E−05 −3.00E−06 7.00E−06 −1.10E−05 −1.00E−05 4.10E−05 6.20 −7.00E−06 3.20E−05 8.00E−05 1.60E−05 −4.00E−06 −7.00E−06 1.80E−05 −1.30E−05 −8.00E−06 4.10E−05 6.22 1.60E−05 2.30E−05 5.40E−05 1.30E−05 1.00E−06 6.00E−06 3.00E−06 −1.60E−05 7.00E−06 5.20E−05 6.23 1.60E−05 4.00E−05 8.00E−05 2.40E−05 3.00E−06 4.00E−06 5.00E−06 −1.00E−05 −1.00E−05 3.50E−05 6.25 1.80E−05 4.00E−05 4.80E−05 7.00E−06 1.20E−05 1.00E−06 7.00E−06 −1.80E−05 −6.00E−06 4.30E−05 6.27 2.10E−05 4.40E−05 7.40E−05 1.80E−05 −1.10E−05 −3.00E−06 −6.00E−06 −2.40E−05 −2.00E−06 4.10E−05 6.28 2.00E−05 3.30E−05 6.60E−05 1.70E−05 2.20E−05 −3.00E−06 2.30E−05 −8.00E−06 −3.00E−06 2.70E−05 6.30 1.70E−05 3.20E−05 8.30E−05 1.30E−05 1.00E−05 4.00E−06 0.00E+00 −1.40E−05 −1.00E−05 4.60E−05 6.32 6.00E−06 4.50E−05 7.10E−05 1.60E−05 3.00E−06 1.00E−05 8.00E−06 −5.00E−06 5.00E−06 3.40E−05 6.33 1.50E−05 3.20E−05 7.60E−05 2.60E−05 6.00E−06 2.00E−06 1.20E−05 −2.70E−05 −1.00E−06 2.30E−05 6.35 3.00E−05 4.30E−05 6.60E−05 2.30E−05 7.00E−06 −3.00E−06 1.60E−05 −2.40E−05 −8.00E−06 3.00E−05 6.37 2.70E−05 4.60E−05 6.20E−05 2.00E−05 1.30E−05 0.00E+00 8.00E−06 −1.40E−05 1.00E−06 3.40E−05 6.38 2.30E−05 4.20E−05 7.50E−05 1.80E−05 −2.00E−06 −2.00E−06 5.00E−06 −2.10E−05 −7.00E−06 4.40E−05 6.40 3.00E−06 2.50E−05 6.60E−05 2.70E−05 6.00E−06 −5.00E−06 1.40E−05 −1.40E−05 −1.50E−05 4.20E−05 6.42 1.80E−05 3.40E−05 8.10E−05 4.00E−06 1.60E−05 −4.00E−06 −4.00E−06 −1.20E−05 −1.20E−05 3.10E−05 6.43 5.00E−06 4.70E−05 6.70E−05 2.50E−05 1.50E−05 1.00E−05 1.50E−05 −7.00E−06 1.00E−06 3.70E−05 6.45 1.20E−05 3.40E−05 7.10E−05 1.40E−05 1.80E−05 6.00E−06 7.00E−06 −1.30E−05 −8.00E−06 3.00E−05 6.47 2.30E−05 4.70E−05 7.30E−05 5.00E−06 5.00E−06 1.10E−05 2.00E−06 −1.10E−05 −1.40E−05 3.80E−05 6.48 2.40E−05 4.20E−05 6.90E−05 1.80E−05 6.00E−06 −2.00E−06 2.00E−06 −5.00E−06 5.00E−06 3.30E−05 6.50 2.50E−05 4.50E−05 6.20E−05 1.60E−05 4.00E−06 8.00E−06 1.70E−05 −7.00E−06 0.00E+00 2.70E−05 6.52 2.10E−05 4.40E−05 7.30E−05 6.00E−06 1.80E−05 2.00E−06 7.00E−06 −1.00E−06 −6.00E−06 3.20E−05 6.53 2.00E−05 4.40E−05 6.00E−05 3.30E−05 4.00E−06 1.40E−05 0.00E+00 −1.80E−05 1.00E−06 3.40E−05 6.55 1.60E−05 5.60E−05 5.20E−05 2.30E−05 7.00E−06 −5.00E−06 1.00E−05 −1.60E−05 2.00E−06 4.10E−05 6.57 1.10E−05 3.00E−05 7.50E−05 1.70E−05 1.00E−06 1.50E−05 1.00E−05 −1.70E−05 −1.00E−06 4.20E−05 6.58 2.10E−05 4.70E−05 6.50E−05 1.00E−05 1.70E−05 6.00E−06 6.00E−06 −1.00E−05 −7.00E−06 4.00E−05 6.60 2.30E−05 4.20E−05 8.10E−05 1.80E−05 1.00E−05 1.40E−05 1.00E−05 −1.30E−05 −6.00E−06 3.80E−05 6.62 1.20E−05 3.60E−05 7.70E−05 2.20E−05 1.10E−05 −1.00E−05 8.00E−06 −3.00E−06 3.00E−06 4.60E−05 6.63 8.00E−06 4.00E−05 6.20E−05 2.30E−05 −2.00E−06 3.00E−06 −5.00E−06 −4.00E−06 2.00E−06 3.80E−05 6.65 1.10E−05 4.20E−05 8.00E−05 2.80E−05 1.70E−05 4.00E−06 1.20E−05 −2.70E−05 4.00E−06 4.80E−05 6.67 1.60E−05 4.80E−05 7.60E−05 2.70E−05 1.00E−06 7.00E−06 1.20E−05 −7.00E−06 −1.10E−05 4.10E−05 6.68 2.20E−05 3.30E−05 8.50E−05 1.50E−05 1.00E−05 −3.00E−06 1.10E−05 −2.70E−05 −2.00E−06 3.10E−05 6.70 1.30E−05 3.30E−05 7.00E−05 2.60E−05 1.10E−05 −1.00E−05 6.00E−06 −8.00E−06 2.00E−06 5.60E−05 6.72 2.00E−05 3.00E−05 8.10E−05 2.50E−05 1.10E−05 2.00E−06 2.40E−05 1.00E−05 7.00E−06 5.10E−05 6.73 2.60E−05 4.80E−05 6.70E−05 1.70E−05 1.20E−05 4.00E−06 1.80E−05 −2.10E−05 −2.00E−06 4.20E−05 6.75 2.70E−05 3.80E−05 7.20E−05 3.10E−05 1.00E−05 2.40E−05 2.00E−06 −2.00E−05 3.00E−06 4.30E−05 6.77 2.50E−05 4.50E−05 8.30E−05 3.10E−05 2.00E−05 5.00E−06 2.10E−05 −2.20E−05 −1.20E−05 3.80E−05 6.78 2.10E−05 3.80E−05 7.70E−05 1.80E−05 1.50E−05 −5.00E−06 7.00E−06 −1.00E−05 −1.10E−05 4.40E−05 6.80 1.40E−05 3.10E−05 6.60E−05 1.40E−05 7.00E−06 1.20E−05 6.00E−06 −1.60E−05 2.00E−06 3.50E−05 6.82 2.60E−05 4.10E−05 7.50E−05 8.00E−06 6.00E−06 1.10E−05 1.10E−05 −1.80E−05 5.00E−06 4.80E−05 6.83 2.80E−05 3.60E−05 6.90E−05 2.20E−05 1.00E−05 1.80E−05 6.00E−06 −1.40E−05 −4.00E−06 5.40E−05 6.85 2.20E−05 3.70E−05 7.60E−05 2.30E−05 1.70E−05 2.00E−05 6.00E−06 −1.40E−05 −1.30E−05 5.50E−05 6.87 3.40E−05 5.30E−05 7.70E−05 3.30E−05 1.60E−05 1.00E−05 1.20E−05 −2.10E−05 7.00E−06 5.50E−05 6.88 3.30E−05 3.80E−05 7.00E−05 1.20E−05 3.50E−05 1.40E−05 6.00E−06 −1.20E−05 −2.00E−06 6.20E−05 6.90 1.80E−05 4.20E−05 8.10E−05 2.20E−05 1.30E−05 1.20E−05 2.00E−06 −4.00E−06 5.00E−06 5.20E−05 6.92 1.60E−05 3.60E−05 7.10E−05 2.60E−05 1.00E−05 2.70E−05 −2.00E−06 −1.00E−05 1.60E−05 5.70E−05 6.93 4.00E−05 3.80E−05 7.60E−05 1.40E−05 4.00E−06 2.50E−05 −2.00E−06 −1.60E−05 2.70E−05 5.70E−05 6.95 3.30E−05 4.40E−05 7.60E−05 1.60E−05 1.70E−05 1.50E−05 7.00E−06 −1.00E−06 −5.00E−06 5.10E−05 6.97 2.80E−05 3.00E−05 6.10E−05 1.70E−05 7.00E−06 2.10E−05 4.00E−06 −3.00E−06 1.30E−05 6.60E−05 6.98 2.50E−05 3.20E−05 7.40E−05 2.80E−05 1.80E−05 3.40E−05 6.00E−06 −1.00E−06 8.00E−06 4.30E−05 7.00 3.30E−05 3.80E−05 7.90E−05 2.70E−05 1.40E−05 2.30E−05 3.00E−06 0.00E+00 1.30E−05 6.50E−05 7.02 3.60E−05 5.30E−05 9.40E−05 6.00E−05 1.30E−05 1.80E−05 1.10E−05 −2.00E−06 1.10E−05 6.60E−05 7.03 3.50E−05 4.10E−05 7.50E−05 2.70E−05 1.70E−05 3.00E−05 1.00E−05 −1.00E−05 1.00E−05 6.20E−05 7.05 3.20E−05 3.70E−05 7.60E−05 3.20E−05 1.70E−05 3.10E−05 1.70E−05 −7.00E−06 1.30E−05 5.50E−05 7.07 3.70E−05 4.60E−05 7.50E−05 3.20E−05 1.10E−05 2.30E−05 4.00E−06 −1.10E−05 1.40E−05 5.40E−05 7.08 3.20E−05 4.20E−05 8.50E−05 3.30E−05 1.70E−05 5.00E−05 −1.00E−06 7.00E−06 4.00E−05 5.80E−05 7.10 2.70E−05 4.00E−05 8.40E−05 3.80E−05 2.10E−05 4.70E−05 −4.00E−06 −2.20E−05 1.70E−05 6.20E−05 7.12 3.30E−05 3.80E−05 7.00E−05 4.70E−05 5.00E−06 4.20E−05 2.00E−06 −7.00E−06 1.80E−05 6.30E−05 7.13 1.80E−05 3.80E−05 7.70E−05 4.40E−05 1.60E−05 5.50E−05 −6.00E−06 −5.00E−06 1.50E−05 6.20E−05 7.15 4.40E−05 4.80E−05 7.70E−05 4.20E−05 2.30E−05 4.70E−05 −2.00E−06 −4.00E−06 2.20E−05 7.20E−05 7.17 5.40E−05 5.60E−05 8.20E−05 4.50E−05 3.20E−05 5.40E−05 1.20E−05 −1.30E−05 4.30E−05 7.00E−05 7.18 2.50E−05 5.00E−05 7.60E−05 5.80E−05 2.40E−05 4.60E−05 1.40E−05 −1.40E−05 5.10E−05 7.00E−05 7.20 3.40E−05 3.60E−05 7.90E−05 5.30E−05 1.00E−05 6.90E−05 1.20E−05 −1.00E−05 2.80E−05 8.70E−05 7.22 3.00E−05 5.10E−05 7.60E−05 5.30E−05 3.10E−05 7.50E−05 −2.00E−06 −1.00E−06 3.30E−05 9.30E−05 7.23 2.50E−05 5.80E−05 7.60E−05 7.30E−05 3.70E−05 8.50E−05 −7.00E−06 −1.30E−05 5.00E−05 9.00E−05 7.25 3.50E−05 5.70E−05 8.00E−05 9.40E−05 1.50E−05 9.60E−05 0.00E+00 1.10E−05 5.60E−05 8.00E−05 7.27 2.30E−05 7.00E−05 9.00E−05 9.30E−05 2.00E−05 9.40E−05 2.10E−05 4.00E−06 5.40E−05 8.70E−05 7.28 3.60E−05 6.20E−05 8.00E−05 8.00E−05 3.20E−05 1.31E−04 8.00E−06 −2.00E−06 5.80E−05 1.14E−04 7.30 5.30E−05 7.00E−05 1.01E−04 1.01E−04 2.10E−05 1.21E−04 −3.00E−06 2.40E−05 6.90E−05 1.13E−04 7.32 4.30E−05 6.50E−05 7.90E−05 1.06E−04 2.20E−05 1.39E−04 −1.10E−05 2.00E−05 8.20E−05 1.19E−04 7.33 5.00E−05 7.50E−05 8.60E−05 1.44E−04 3.10E−05 1.44E−04 1.20E−05 6.00E−06 8.60E−05 1.15E−04 7.35 4.10E−05 8.20E−05 7.10E−05 1.31E−04 2.50E−05 1.69E−04 1.50E−05 2.50E−05 9.10E−05 1.26E−04 7.37 4.10E−05 7.10E−05 8.50E−05 1.51E−04 2.60E−05 1.98E−04 −3.00E−06 2.20E−05 1.15E−04 1.35E−04 7.38 3.70E−05 8.30E−05 8.50E−05 1.74E−04 1.70E−05 2.11E−04 2.00E−06 3.50E−05 1.23E−04 1.59E−04 7.40 4.80E−05 9.50E−05 8.30E−05 1.92E−04 1.80E−05 2.24E−04 1.40E−05 1.50E−05 1.35E−04 1.59E−04 7.42 5.50E−05 1.04E−04 9.40E−05 2.07E−04 3.30E−05 2.59E−04 1.20E−05 3.10E−05 1.62E−04 1.76E−04 7.43 3.30E−05 1.11E−04 9.10E−05 2.40E−04 1.70E−05 2.82E−04 1.00E−05 2.30E−05 1.74E−04 1.92E−04 7.45 2.30E−05 1.14E−04 9.90E−05 2.48E−04 2.80E−05 3.18E−04 1.50E−05 5.00E−05 1.86E−04 2.02E−04 7.47 2.70E−05 1.31E−04 9.20E−05 2.63E−04 2.40E−05 3.33E−04 1.50E−05 5.10E−05 2.12E−04 2.34E−04 7.48 4.60E−05 1.45E−04 1.10E−04 3.00E−04 2.80E−05 3.75E−04 1.00E−05 5.50E−05 2.29E−04 2.41E−04 7.50 4.00E−05 1.46E−04 8.60E−05 3.46E−04 3.20E−05 4.00E−04 7.00E−06 5.40E−05 2.43E−04 2.64E−04 7.52 4.30E−05 1.64E−04 8.60E−05 3.68E−04 2.20E−05 4.37E−04 8.00E−06 8.40E−05 2.64E−04 2.92E−04 7.53 5.10E−05 1.79E−04 8.20E−05 3.80E−04 1.80E−05 4.73E−04 8.00E−06 9.20E−05 3.12E−04 3.18E−04 7.55 4.80E−05 1.86E−04 9.70E−05 4.24E−04 3.10E−05 5.15E−04 1.30E−05 1.09E−04 3.25E−04 3.57E−04 7.57 3.70E−05 1.93E−04 1.01E−04 4.75E−04 1.70E−05 5.44E−04 3.40E−05 1.10E−04 3.69E−04 3.79E−04 7.58 3.60E−05 2.21E−04 1.16E−04 5.29E−04 3.50E−05 6.16E−04 2.80E−05 1.29E−04 3.94E−04 4.00E−04 7.60 4.40E−05 2.39E−04 1.04E−04 5.69E−04 3.40E−05 6.60E−04 2.30E−05 1.69E−04 4.26E−04 4.35E−04 7.62 5.20E−05 2.62E−04 1.17E−04 6.03E−04 5.00E−06 7.52E−04 2.10E−05 1.68E−04 4.74E−04 4.70E−04 7.63 6.30E−05 2.82E−04 1.05E−04 6.71E−04 3.20E−05 8.04E−04 1.60E−05 1.83E−04 5.19E−04 5.18E−04 7.65 2.60E−05 3.06E−04 1.07E−04 7.42E−04 3.00E−05 8.93E−04 4.70E−05 1.99E−04 5.94E−04 5.65E−04 7.67 3.10E−05 3.25E−04 1.15E−04 8.13E−04 2.20E−05 9.87E−04 2.60E−05 2.28E−04 6.40E−04 6.02E−04 7.68 4.10E−05 3.45E−04 1.22E−04 8.99E−04 2.40E−05 1.10E−03 3.20E−05 2.71E−04 7.25E−04 6.81E−04 7.70 4.80E−05 3.84E−04 1.11E−04 9.95E−04 3.50E−05 1.21E−03 3.50E−05 3.00E−04 8.04E−04 7.36E−04 7.72 4.00E−05 4.27E−04 1.39E−04 1.11E−03 2.50E−05 1.35E−03 3.60E−05 3.26E−04 9.01E−04 8.00E−04 7.73 3.70E−05 4.69E−04 1.31E−04 1.22E−03 3.10E−05 1.49E−03 3.70E−05 3.48E−04 9.98E−04 9.03E−04 7.75 4.30E−05 5.12E−04 1.28E−04 1.35E−03 4.50E−05 1.62E−03 3.60E−05 3.94E−04 1.12E−03 1.01E−03 7.77 4.50E−05 5.73E−04 1.29E−04 1.47E−03 3.20E−05 1.79E−03 4.30E−05 4.46E−04 1.23E−03 1.10E−03 7.78 4.30E−05 6.21E−04 1.56E−04 1.62E−03 4.40E−05 1.95E−03 2.80E−05 5.04E−04 1.36E−03 1.23E−03 7.80 4.40E−05 6.95E−04 1.43E−04 1.80E−03 3.30E−05 2.10E−03 4.30E−05 5.59E−04 1.50E−03 1.35E−03 7.82 5.20E−05 7.66E−04 1.58E−04 1.94E−03 4.00E−05 2.25E−03 6.00E−05 6.26E−04 1.63E−03 1.49E−03 7.83 4.80E−05 8.43E−04 1.62E−04 2.10E−03 3.10E−05 2.42E−03 6.30E−05 7.29E−04 1.78E−03 1.63E−03 7.85 6.00E−05 9.28E−04 1.70E−04 2.25E−03 4.60E−05 2.59E−03 7.40E−05 8.02E−04 1.94E−03 1.77E−03 7.87 4.00E−05 1.02E−03 1.85E−04 2.40E−03 5.20E−05 2.74E−03 5.60E−05 8.84E−04 2.07E−03 1.93E−03 7.88 4.40E−05 1.13E−03 2.02E−04 2.58E−03 4.60E−05 2.89E−03 5.30E−05 9.70E−04 2.22E−03 2.09E−03 7.90 5.60E−05 1.22E−03 1.93E−04 2.70E−03 6.60E−05 3.01E−03 7.90E−05 1.10E−03 2.35E−03 2.23E−03 7.92 5.10E−05 1.32E−03 2.30E−04 2.85E−03 6.90E−05 3.14E−03 8.40E−05 1.20E−03 2.47E−03 2.38E−03 7.93 4.40E−05 1.44E−03 2.35E−04 2.96E−03 6.50E−05 3.23E−03 9.50E−05 1.33E−03 2.58E−03 2.52E−03 7.95 6.00E−05 1.57E−03 2.47E−04 3.10E−03 7.00E−05 3.33E−03 9.50E−05 1.44E−03 2.71E−03 2.66E−03 7.97 5.40E−05 1.64E−03 2.62E−04 3.18E−03 1.01E−04 3.43E−03 1.04E−04 1.57E−03 2.82E−03 2.77E−03 7.98 5.40E−05 1.74E−03 2.92E−04 3.28E−03 1.12E−04 3.49E−03 1.26E−04 1.69E−03 2.92E−03 2.89E−03 8.00 6.10E−05 1.84E−03 3.10E−04 3.36E−03 1.22E−04 3.59E−03 1.29E−04 1.83E−03 3.00E−03 3.00E−03 8.02 5.20E−05 1.92E−03 3.45E−04 3.43E−03 1.42E−04 3.67E−03 1.30E−04 1.96E−03 3.11E−03 3.11E−03 8.03 5.00E−05 2.02E−03 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3.12E−03 5.78E−03 4.97E−03 8.22 5.30E−05 2.95E−03 7.71E−04 6.65E−03 1.43E−03 8.06E−03 4.39E−04 3.26E−03 6.38E−03 5.41E−03 8.23 6.30E−05 3.15E−03 8.21E−04 7.37E−03 1.76E−03 8.95E−03 4.74E−04 3.47E−03 7.08E−03 5.91E−03 8.25 6.00E−05 3.38E−03 8.80E−04 8.20E−03 2.17E−03 9.97E−03 5.56E−04 3.69E−03 7.87E−03 6.49E−03 8.27 6.20E−05 3.69E−03 9.03E−04 9.12E−03 2.66E−03 1.11E−02 5.47E−04 3.99E−03 8.76E−03 7.19E−03 8.28 7.10E−05 4.02E−03 9.68E−04 1.01E−02 3.25E−03 1.22E−02 6.06E−04 4.31E−03 9.71E−03 7.99E−03 8.30 6.50E−05 4.42E−03 1.01E−03 1.12E−02 3.91E−03 1.35E−02 6.33E−04 4.69E−03 1.07E−02 8.84E−03 8.32 6.00E−05 4.89E−03 1.05E−03 1.24E−02 4.68E−03 1.48E−02 6.86E−04 5.15E−03 1.19E−02 9.83E−03 8.33 7.00E−05 5.45E−03 1.12E−03 1.37E−02 5.53E−03 1.62E−02 7.53E−04 5.67E−03 1.30E−02 1.09E−02 8.35 6.20E−05 6.08E−03 1.20E−03 1.50E−02 6.49E−03 1.75E−02 7.94E−04 6.28E−03 1.42E−02 1.20E−02 8.37 6.50E−05 6.74E−03 1.25E−03 1.63E−02 7.48E−03 1.89E−02 8.62E−04 6.97E−03 1.54E−02 1.32E−02 8.38 5.60E−05 7.49E−03 1.33E−03 1.76E−02 8.52E−03 2.02E−02 9.40E−04 7.76E−03 1.67E−02 1.44E−02 8.40 6.70E−05 8.33E−03 1.44E−03 1.89E−02 9.59E−03 2.14E−02 9.87E−04 8.60E−03 1.79E−02 1.57E−02 8.42 8.40E−05 9.21E−03 1.53E−03 2.02E−02 1.07E−02 2.26E−02 1.05E−03 9.53E−03 1.90E−02 1.69E−02 8.43 6.40E−05 1.01E−02 1.67E−03 2.14E−02 1.18E−02 2.37E−02 1.15E−03 1.05E−02 2.01E−02 1.81E−02 8.45 6.30E−05 1.11E−02 1.82E−03 2.25E−02 1.29E−02 2.46E−02 1.24E−03 1.15E−02 2.11E−02 1.93E−02 8.47 6.00E−05 1.21E−02 1.99E−03 2.35E−02 1.39E−02 2.54E−02 1.35E−03 1.26E−02 2.20E−02 2.05E−02 8.48 5.50E−05 1.31E−02 2.17E−03 2.44E−02 1.48E−02 2.61E−02 1.46E−03 1.38E−02 2.28E−02 2.15E−02 8.50 6.20E−05 1.40E−02 2.40E−03 2.52E−02 1.56E−02 2.66E−02 1.61E−03 1.49E−02 2.34E−02 2.24E−02 8.52 4.70E−05 1.50E−02 2.66E−03 2.58E−02 1.63E−02 2.69E−02 1.74E−03 1.60E−02 2.39E−02 2.33E−02 8.53 5.70E−05 1.59E−02 2.94E−03 2.63E−02 1.69E−02 2.71E−02 1.91E−03 1.71E−02 2.43E−02 2.40E−02 8.55 5.60E−05 1.67E−02 3.27E−03 2.65E−02 1.74E−02 2.70E−02 2.12E−03 1.81E−02 2.45E−02 2.46E−02 8.57 6.00E−05 1.75E−02 3.63E−03 2.67E−02 1.78E−02 2.69E−02 2.34E−03 1.91E−02 2.46E−02 2.50E−02 8.58 6.10E−05 1.82E−02 4.01E−03 2.66E−02 1.81E−02 2.65E−02 2.58E−03 2.01E−02 2.45E−02 2.52E−02 8.60 6.60E−05 1.88E−02 4.43E−03 2.65E−02 1.82E−02 2.61E−02 2.87E−03 2.08E−02 2.43E−02 2.53E−02 8.62 5.10E−05 1.93E−02 4.90E−03 2.61E−02 1.83E−02 2.55E−02 3.18E−03 2.16E−02 2.40E−02 2.53E−02 8.63 6.00E−05 1.97E−02 5.35E−03 2.57E−02 1.82E−02 2.48E−02 3.52E−03 2.22E−02 2.36E−02 2.51E−02 8.65 5.70E−05 2.00E−02 5.88E−03 2.51E−02 1.81E−02 2.40E−02 3.90E−03 2.27E−02 2.30E−02 2.48E−02 8.67 5.70E−05 2.02E−02 6.39E−03 2.44E−02 1.79E−02 2.32E−02 4.31E−03 2.31E−02 2.24E−02 2.44E−02 8.68 5.50E−05 2.02E−02 6.91E−03 2.37E−02 1.76E−02 2.23E−02 4.75E−03 2.33E−02 2.17E−02 2.39E−02 8.70 5.60E−05 2.02E−02 7.45E−03 2.29E−02 1.72E−02 2.14E−02 5.22E−03 2.34E−02 2.10E−02 2.33E−02 8.72 6.10E−05 2.00E−02 8.01E−03 2.20E−02 1.69E−02 2.05E−02 5.72E−03 2.34E−02 2.03E−02 2.26E−02 8.73 5.80E−05 1.98E−02 8.54E−03 2.11E−02 1.65E−02 1.96E−02 6.24E−03 2.33E−02 1.95E−02 2.18E−02 8.75 6.60E−05 1.94E−02 9.07E−03 2.02E−02 1.61E−02 1.87E−02 6.80E−03 2.31E−02 1.87E−02 2.11E−02 8.77 6.10E−05 1.90E−02 9.58E−03 1.94E−02 1.57E−02 1.78E−02 7.36E−03 2.27E−02 1.80E−02 2.03E−02 8.78 5.10E−05 1.85E−02 1.01E−02 1.85E−02 1.53E−02 1.70E−02 7.93E−03 2.23E−02 1.72E−02 1.95E−02 8.80 6.90E−05 1.79E−02 1.05E−02 1.77E−02 1.49E−02 1.63E−02 8.51E−03 2.18E−02 1.65E−02 1.87E−02 8.82 4.70E−05 1.73E−02 1.09E−02 1.69E−02 1.46E−02 1.56E−02 9.08E−03 2.12E−02 1.59E−02 1.79E−02 8.83 5.30E−05 1.67E−02 1.13E−02 1.63E−02 1.43E−02 1.50E−02 9.66E−03 2.06E−02 1.53E−02 1.72E−02 8.85 5.70E−05 1.61E−02 1.17E−02 1.56E−02 1.40E−02 1.45E−02 1.02E−02 1.99E−02 1.48E−02 1.65E−02 8.87 5.60E−05 1.54E−02 1.20E−02 1.51E−02 1.38E−02 1.41E−02 1.08E−02 1.92E−02 1.43E−02 1.58E−02 8.88 4.40E−05 1.47E−02 1.22E−02 1.46E−02 1.36E−02 1.38E−02 1.13E−02 1.85E−02 1.39E−02 1.53E−02 8.90 5.70E−05 1.41E−02 1.24E−02 1.42E−02 1.35E−02 1.35E−02 1.18E−02 1.78E−02 1.36E−02 1.48E−02 8.92 7.10E−05 1.34E−02 1.26E−02 1.39E−02 1.34E−02 1.34E−02 1.22E−02 1.71E−02 1.34E−02 1.43E−02 8.93 5.70E−05 1.28E−02 1.27E−02 1.37E−02 1.35E−02 1.34E−02 1.27E−02 1.64E−02 1.33E−02 1.40E−02 8.95 6.10E−05 1.22E−02 1.27E−02 1.36E−02 1.36E−02 1.36E−02 1.30E−02 1.58E−02 1.33E−02 1.37E−02 8.97 6.40E−05 1.17E−02 1.27E−02 1.37E−02 1.39E−02 1.39E−02 1.34E−02 1.52E−02 1.34E−02 1.36E−02 8.98 6.20E−05 1.13E−02 1.27E−02 1.39E−02 1.42E−02 1.44E−02 1.37E−02 1.47E−02 1.38E−02 1.35E−02 9.00 6.50E−05 1.09E−02 1.26E−02 1.43E−02 1.48E−02 1.52E−02 1.39E−02 1.42E−02 1.43E−02 1.36E−02 9.02 7.00E−05 1.06E−02 1.25E−02 1.49E−02 1.55E−02 1.61E−02 1.41E−02 1.38E−02 1.51E−02 1.39E−02 9.03 7.20E−05 1.03E−02 1.23E−02 1.57E−02 1.66E−02 1.75E−02 1.43E−02 1.35E−02 1.61E−02 1.43E−02 9.05 5.30E−05 1.02E−02 1.21E−02 1.68E−02 1.78E−02 1.92E−02 1.44E−02 1.34E−02 1.75E−02 1.50E−02 9.07 7.50E−05 1.03E−02 1.19E−02 1.83E−02 1.96E−02 2.15E−02 1.45E−02 1.33E−02 1.93E−02 1.60E−02 9.08 6.10E−05 1.05E−02 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3.48E−02 8.84E−02 6.54E−02 9.27 6.50E−05 3.87E−02 1.13E−02 9.33E−02 9.96E−02 1.14E−01 1.50E−02 4.03E−02 1.00E−01 7.54E−02 9.28 6.10E−05 4.54E−02 1.19E−02 1.06E−01 1.13E−01 1.29E−01 1.53E−02 4.68E−02 1.13E−01 8.64E−02 9.30 6.50E−05 5.28E−02 1.27E−02 1.20E−01 1.26E−01 1.45E−01 1.58E−02 5.41E−02 1.27E−01 9.83E−02 9.32 6.60E−05 6.12E−02 1.39E−02 1.35E−01 1.41E−01 1.61E−01 1.65E−02 6.23E−02 1.41E−01 1.11E−01 9.33 7.20E−05 7.05E−02 1.53E−02 1.51E−01 1.56E−01 1.78E−01 1.73E−02 7.15E−02 1.56E−01 1.25E−01 9.35 5.70E−05 8.08E−02 1.71E−02 1.67E−01 1.71E−01 1.94E−01 1.85E−02 8.15E−02 1.71E−01 1.39E−01 9.37 6.70E−05 9.19E−02 1.94E−02 1.83E−01 1.86E−01 2.12E−01 1.98E−02 9.25E−02 1.86E−01 1.54E−01 9.38 8.50E−05 1.04E−01 2.21E−02 2.00E−01 2.01E−01 2.29E−01 2.16E−02 1.04E−01 2.01E−01 1.69E−01 9.40 8.90E−05 1.16E−01 2.54E−02 2.17E−01 2.15E−01 2.45E−01 2.37E−02 1.17E−01 2.15E−01 1.85E−01 9.42 7.70E−05 1.29E−01 2.92E−02 2.34E−01 2.29E−01 2.61E−01 2.62E−02 1.30E−01 2.28E−01 2.00E−01 9.43 7.30E−05 1.43E−01 3.36E−02 2.50E−01 2.41E−01 2.75E−01 2.93E−02 1.43E−01 2.41E−01 2.15E−01 9.45 7.90E−05 1.56E−01 3.87E−02 2.65E−01 2.52E−01 2.89E−01 3.28E−02 1.57E−01 2.52E−01 2.29E−01 9.47 6.70E−05 1.70E−01 4.45E−02 2.79E−01 2.62E−01 3.01E−01 3.69E−02 1.71E−01 2.62E−01 2.43E−01 9.48 6.90E−05 1.83E−01 5.09E−02 2.91E−01 2.69E−01 3.10E−01 4.16E−02 1.85E−01 2.70E−01 2.55E−01 9.50 7.00E−05 1.96E−01 5.80E−02 3.02E−01 2.75E−01 3.18E−01 4.70E−02 1.98E−01 2.76E−01 2.66E−01 9.52 7.10E−05 2.09E−01 6.56E−02 3.11E−01 2.79E−01 3.24E−01 5.30E−02 2.11E−01 2.81E−01 2.75E−01 9.53 7.00E−05 2.21E−01 7.39E−02 3.18E−01 2.80E−01 3.27E−01 5.96E−02 2.24E−01 2.83E−01 2.82E−01 9.55 6.50E−05 2.32E−01 8.29E−02 3.23E−01 2.79E−01 3.28E−01 6.69E−02 2.35E−01 2.83E−01 2.87E−01 9.57 4.80E−05 2.41E−01 9.23E−02 3.25E−01 2.76E−01 3.26E−01 7.49E−02 2.46E−01 2.81E−01 2.90E−01 9.58 6.20E−05 2.50E−01 1.02E−01 3.25E−01 2.71E−01 3.22E−01 8.35E−02 2.55E−01 2.77E−01 2.91E−01 9.60 8.30E−05 2.57E−01 1.12E−01 3.22E−01 2.64E−01 3.15E−01 9.26E−02 2.63E−01 2.71E−01 2.90E−01 9.62 6.40E−05 2.62E−01 1.23E−01 3.17E−01 2.55E−01 3.06E−01 1.02E−01 2.69E−01 2.63E−01 2.87E−01 9.63 6.40E−05 2.66E−01 1.33E−01 3.10E−01 2.45E−01 2.96E−01 1.12E−01 2.74E−01 2.54E−01 2.82E−01 9.65 7.70E−05 2.67E−01 1.44E−01 3.00E−01 2.34E−01 2.83E−01 1.23E−01 2.77E−01 2.43E−01 2.74E−01 9.67 7.90E−05 2.68E−01 1.54E−01 2.89E−01 2.21E−01 2.69E−01 1.33E−01 2.78E−01 2.32E−01 2.65E−01 9.68 7.10E−05 2.66E−01 1.65E−01 2.76E−01 2.08E−01 2.55E−01 1.44E−01 2.77E−01 2.19E−01 2.55E−01 9.70 6.60E−05 2.63E−01 1.74E−01 2.62E−01 1.95E−01 2.39E−01 1.55E−01 2.75E−01 2.06E−01 2.43E−01 9.72 6.70E−05 2.58E−01 1.84E−01 2.47E−01 1.81E−01 2.23E−01 1.66E−01 2.71E−01 1.93E−01 2.31E−01 9.73 9.40E−05 2.51E−01 1.93E−01 2.32E−01 1.68E−01 2.07E−01 1.76E−01 2.65E−01 1.79E−01 2.18E−01 9.75 8.60E−05 2.43E−01 2.01E−01 2.16E−01 1.54E−01 1.91E−01 1.87E−01 2.58E−01 1.66E−01 2.04E−01 9.77 7.90E−05 2.35E−01 2.09E−01 2.00E−01 1.42E−01 1.75E−01 1.96E−01 2.50E−01 1.53E−01 1.90E−01 9.78 8.60E−05 2.25E−01 2.15E−01 1.85E−01 1.29E−01 1.60E−01 2.06E−01 2.40E−01 1.41E−01 1.77E−01 9.80 6.70E−05 2.15E−01 2.21E−01 1.70E−01 1.18E−01 1.45E−01 2.15E−01 2.30E−01 1.29E−01 1.63E−01 9.82 6.90E−05 2.04E−01 2.25E−01 1.55E−01 1.07E−01 1.32E−01 2.23E−01 2.19E−01 1.18E−01 1.50E−01 9.83 7.20E−05 1.92E−01 2.29E−01 1.41E−01 9.70E−02 1.19E−01 2.30E−01 2.08E−01 1.07E−01 1.37E−01 9.85 8.60E−05 1.81E−01 2.31E−01 1.28E−01 8.79E−02 1.08E−01 2.36E−01 1.96E−01 9.71E−02 1.26E−01 9.87 5.80E−05 1.69E−01 2.33E−01 1.16E−01 7.95E−02 9.70E−02 2.42E−01 1.84E−01 8.81E−02 1.15E−01 9.88 9.10E−05 1.58E−01 2.33E−01 1.05E−01 7.17E−02 8.73E−02 2.46E−01 1.73E−01 7.99E−02 1.04E−01 9.90 8.00E−05 1.47E−01 2.33E−01 9.47E−02 6.47E−02 7.85E−02 2.50E−01 1.61E−01 7.22E−02 9.44E−02 9.92 7.30E−05 1.36E−01 2.31E−01 8.53E−02 5.85E−02 7.06E−02 2.52E−01 1.50E−01 6.53E−02 8.55E−02 9.93 7.10E−05 1.25E−01 2.28E−01 7.69E−02 5.29E−02 6.34E−02 2.53E−01 1.39E−01 5.90E−02 7.74E−02 9.95 6.90E−05 1.16E−01 2.25E−01 6.91E−02 4.78E−02 5.69E−02 2.54E−01 1.28E−01 5.34E−02 7.00E−02 9.97 8.60E−05 1.06E−01 2.21E−01 6.22E−02 4.33E−02 5.12E−02 2.53E−01 1.18E−01 4.83E−02 6.33E−02 9.98 7.60E−05 9.74E−02 2.16E−01 5.60E−02 3.92E−02 4.62E−02 2.51E−01 1.09E−01 4.37E−02 5.72E−02 10.00 6.60E−05 8.92E−02 2.10E−01 5.05E−02 3.57E−02 4.17E−02 2.49E−01 1.00E−01 3.97E−02 5.17E−02 10.02 8.10E−05 8.16E−02 2.04E−01 4.56E−02 3.25E−02 3.77E−02 2.45E−01 9.19E−02 3.61E−02 4.68E−02 10.03 8.00E−05 7.46E−02 1.97E−01 4.12E−02 2.97E−02 3.42E−02 2.41E−01 8.42E−02 3.29E−02 4.25E−02 10.05 7.90E−05 6.81E−02 1.90E−01 3.73E−02 2.72E−02 3.12E−02 2.36E−01 7.71E−02 3.00E−02 3.86E−02 10.07 6.30E−05 6.21E−02 1.82E−01 3.39E−02 2.50E−02 2.84E−02 2.31E−01 7.06E−02 2.74E−02 3.51E−02 10.08 6.50E−05 5.66E−02 1.75E−01 3.09E−02 2.31E−02 2.61E−02 2.25E−01 6.46E−02 2.52E−02 3.20E−02 10.10 6.90E−05 5.17E−02 1.67E−01 2.83E−02 2.14E−02 2.40E−02 2.19E−01 5.90E−02 2.32E−02 2.92E−02 10.12 8.10E−05 4.72E−02 1.59E−01 2.59E−02 1.99E−02 2.21E−02 2.12E−01 5.40E−02 2.14E−02 2.68E−02 10.13 7.40E−05 4.31E−02 1.51E−01 2.39E−02 1.85E−02 2.05E−02 2.04E−01 4.94E−02 1.99E−02 2.46E−02 10.15 7.20E−05 3.94E−02 1.44E−01 2.20E−02 1.73E−02 1.91E−02 1.97E−01 4.53E−02 1.84E−02 2.27E−02 10.17 6.60E−05 3.60E−02 1.36E−01 2.04E−02 1.63E−02 1.79E−02 1.89E−01 4.15E−02 1.72E−02 2.11E−02 10.18 7.10E−05 3.30E−02 1.29E−01 1.91E−02 1.53E−02 1.68E−02 1.81E−01 3.81E−02 1.61E−02 1.96E−02 10.20 7.90E−05 3.04E−02 1.21E−01 1.78E−02 1.45E−02 1.58E−02 1.72E−01 3.50E−02 1.52E−02 1.83E−02 10.22 6.30E−05 2.79E−02 1.14E−01 1.67E−02 1.37E−02 1.50E−02 1.64E−01 3.22E−02 1.43E−02 1.70E−02 10.23 6.50E−05 2.57E−02 1.07E−01 1.57E−02 1.31E−02 1.42E−02 1.57E−01 2.97E−02 1.35E−02 1.60E−02 10.25 8.50E−05 2.37E−02 1.01E−01 1.49E−02 1.25E−02 1.35E−02 1.49E−01 2.74E−02 1.28E−02 1.51E−02 10.27 7.70E−05 2.20E−02 9.45E−02 1.42E−02 2.97E−02 3.42E−02 1.42E−01 2.53E−02 1.22E−02 1.42E−02 10.28 5.40E−05 2.04E−02 8.85E−02 1.35E−02 1.14E−02 1.24E−02 1.35E−01 2.35E−02 1.16E−02 1.35E−02 10.30 8.30E−05 1.90E−02 8.29E−02 1.28E−02 1.09E−02 1.19E−02 1.28E−01 2.18E−02 1.11E−02 1.28E−02 10.32 6.70E−05 1.77E−02 7.76E−02 1.23E−02 1.05E−02 1.14E−02 1.22E−01 2.04E−02 1.07E−02 1.22E−02 10.33 7.30E−05 1.65E−02 7.27E−02 1.18E−02 1.01E−02 1.10E−02 1.16E−01 1.90E−02 1.02E−02 1.17E−02 10.35 6.40E−05 1.55E−02 6.82E−02 1.13E−02 9.79E−03 1.06E−02 1.09E−01 1.78E−02 9.86E−03 1.12E−02 10.37 8.10E−05 1.47E−02 6.38E−02 1.09E−02 9.42E−03 1.03E−02 1.03E−01 1.68E−02 9.51E−03 1.07E−02 10.38 7.10E−05 1.38E−02 5.97E−02 1.05E−02 9.10E−03 9.97E−03 9.67E−02 1.58E−02 9.16E−03 1.03E−02 10.40 6.60E−05 1.31E−02 5.59E−02 1.02E−02 8.84E−03 9.64E−03 9.06E−02 1.49E−02 8.86E−03 9.93E−03 10.42 7.70E−05 1.24E−02 5.22E−02 9.82E−03 8.54E−03 9.39E−03 8.47E−02 1.41E−02 8.58E−03 9.58E−03 10.43 6.70E−05 1.18E−02 4.87E−02 9.53E−03 8.32E−03 9.09E−03 7.90E−02 1.34E−02 8.31E−03 9.30E−03 10.45 5.80E−05 1.12E−02 4.53E−02 9.23E−03 8.02E−03 8.82E−03 7.38E−02 1.27E−02 8.05E−03 8.93E−03 10.47 7.00E−05 1.07E−02 4.22E−02 8.94E−03 7.80E−03 8.60E−03 6.91E−02 1.21E−02 7.80E−03 8.65E−03 10.48 6.30E−05 1.02E−02 3.92E−02 8.68E−03 7.58E−03 8.31E−03 6.48E−02 1.16E−02 7.56E−03 8.39E−03 10.50 6.00E−05 9.80E−03 3.65E−02 8.42E−03 7.35E−03 8.12E−03 6.11E−02 1.10E−02 7.36E−03 8.13E−03 10.52 5.70E−05 9.39E−03 3.40E−02 8.20E−03 7.15E−03 7.89E−03 5.77E−02 1.06E−02 7.15E−03 7.87E−03 10.53 6.90E−05 9.04E−03 3.17E−02 8.00E−03 6.96E−03 7.68E−03 5.47E−02 1.01E−02 6.98E−03 7.63E−03 10.55 6.50E−05 8.70E−03 2.98E−02 7.73E−03 6.78E−03 7.49E−03 5.20E−02 9.71E−03 6.75E−03 7.42E−03 10.57 7.40E−05 8.38E−03 2.80E−02 7.52E−03 6.60E−03 7.29E−03 4.94E−02 9.39E−03 6.57E−03 7.22E−03 10.58 6.30E−05 8.07E−03 2.63E−02 7.31E−03 6.41E−03 7.10E−03 4.70E−02 9.03E−03 6.40E−03 7.03E−03 10.60 6.20E−05 7.80E−03 2.49E−02 7.11E−03 6.25E−03 6.91E−03 4.45E−02 8.71E−03 6.24E−03 6.80E−03 10.62 7.10E−05 7.58E−03 2.35E−02 6.93E−03 6.09E−03 6.74E−03 4.20E−02 8.42E−03 6.05E−03 6.61E−03 10.63 5.80E−05 7.34E−03 2.23E−02 6.73E−03 5.93E−03 6.58E−03 3.95E−02 8.15E−03 5.90E−03 6.46E−03 10.65 5.50E−05 7.11E−03 2.11E−02 6.56E−03 5.78E−03 6.42E−03 3.70E−02 7.91E−03 5.75E−03 6.29E−03 10.67 6.10E−05 6.89E−03 2.00E−02 6.40E−03 5.65E−03 6.24E−03 3.45E−02 7.66E−03 5.60E−03 6.16E−03 10.68 5.50E−05 6.68E−03 1.89E−02 6.24E−03 5.49E−03 6.10E−03 3.20E−02 7.40E−03 5.47E−03 5.95E−03 10.70 6.30E−05 6.51E−03 1.79E−02 6.06E−03 5.35E−03 5.95E−03 2.97E−02 7.18E−03 5.30E−03 5.81E−03 10.72 8.40E−05 6.30E−03 1.69E−02 5.89E−03 5.21E−03 5.78E−03 2.76E−02 6.96E−03 5.16E−03 5.67E−03 10.73 7.00E−05 6.12E−03 1.59E−02 5.74E−03 5.12E−03 5.67E−03 2.57E−02 6.78E−03 5.02E−03 5.51E−03 10.75 6.90E−05 5.93E−03 1.49E−02 5.63E−03 4.98E−03 5.50E−03 2.38E−02 6.57E−03 4.92E−03 5.36E−03 10.77 7.20E−05 5.78E−03 1.41E−02 5.48E−03 4.84E−03 5.35E−03 2.23E−02 6.38E−03 4.78E−03 5.22E−03 10.78 5.80E−05 5.63E−03 1.32E−02 5.31E−03 4.72E−03 5.21E−03 2.11E−02 6.18E−03 4.64E−03 5.09E−03 10.80 7.60E−05 5.46E−03 1.25E−02 5.16E−03 4.60E−03 5.08E−03 2.00E−02 6.00E−03 4.52E−03 4.94E−03 10.82 7.60E−05 5.31E−03 1.19E−02 5.01E−03 4.49E−03 4.95E−03 1.90E−02 5.85E−03 4.39E−03 4.83E−03 10.83 5.80E−05 5.15E−03 1.13E−02 4.91E−03 4.40E−03 4.83E−03 1.83E−02 5.69E−03 4.29E−03 4.68E−03 10.85 6.50E−05 5.00E−03 1.08E−02 4.78E−03 4.26E−03 4.70E−03 1.77E−02 5.53E−03 4.19E−03 4.57E−03 10.87 7.20E−05 4.86E−03 1.03E−02 4.64E−03 4.14E−03 4.56E−03 1.71E−02 5.37E−03 4.05E−03 4.45E−03 10.88 6.90E−05 4.72E−03 9.95E−03 4.51E−03 4.02E−03 4.44E−03 1.66E−02 5.22E−03 3.94E−03 4.35E−03 10.90 7.70E−05 4.59E−03 9.60E−03 4.39E−03 3.91E−03 4.33E−03 1.60E−02 5.09E−03 3.85E−03 4.24E−03 10.92 7.30E−05 4.45E−03 9.30E−03 4.26E−03 3.83E−03 4.20E−03 1.55E−02 4.94E−03 3.76E−03 4.11E−03 10.93 7.40E−05 4.35E−03 8.99E−03 4.15E−03 3.71E−03 4.09E−03 1.49E−02 4.82E−03 3.63E−03 4.00E−03 10.95 9.30E−05 4.24E−03 8.73E−03 4.04E−03 3.62E−03 3.99E−03 1.43E−02 4.67E−03 3.52E−03 3.90E−03 10.97 8.10E−05 4.11E−03 8.46E−03 3.92E−03 3.52E−03 3.86E−03 1.37E−02 4.55E−03 3.42E−03 3.78E−03 10.98 1.40E−04 4.00E−03 8.20E−03 3.80E−03 3.44E−03 3.76E−03 1.30E−02 4.45E−03 3.34E−03 3.67E−03 11.00 8.40E−05 3.89E−03 7.93E−03 3.71E−03 3.32E−03 3.67E−03 1.24E−02 4.33E−03 3.24E−03 3.59E−03 11.02 9.60E−05 3.80E−03 7.67E−03 3.60E−03 3.23E−03 3.57E−03 1.17E−02 4.20E−03 3.16E−03 3.48E−03 11.03 9.90E−05 3.69E−03 7.41E−03 3.50E−03 3.20E−03 3.45E−03 1.11E−02 4.09E−03 3.06E−03 3.40E−03 11.05 1.01E−04 3.59E−03 7.15E−03 3.42E−03 3.07E−03 3.36E−03 1.05E−02 3.99E−03 2.99E−03 3.30E−03 11.07 1.06E−04 3.48E−03 6.87E−03 3.31E−03 2.98E−03 3.26E−03 9.82E−03 3.87E−03 2.88E−03 3.21E−03 11.08 1.20E−04 3.37E−03 6.63E−03 3.20E−03 2.89E−03 3.17E−03 9.29E−03 3.77E−03 2.81E−03 3.12E−03 11.10 1.11E−04 3.31E−03 6.36E−03 3.12E−03 2.81E−03 3.08E−03 8.81E−03 3.65E−03 2.72E−03 3.03E−03 11.12 1.48E−04 3.20E−03 6.12E−03 3.02E−03 2.74E−03 2.99E−03 8.38E−03 3.55E−03 2.63E−03 2.93E−03 11.13 1.54E−04 3.11E−03 5.90E−03 2.94E−03 2.68E−03 2.89E−03 7.99E−03 3.44E−03 2.58E−03 2.85E−03 11.15 1.66E−04 3.01E−03 5.68E−03 2.86E−03 2.59E−03 2.81E−03 7.59E−03 3.35E−03 2.51E−03 2.78E−03 11.17 1.69E−04 2.92E−03 5.49E−03 2.77E−03 2.52E−03 2.74E−03 7.31E−03 3.24E−03 2.42E−03 2.71E−03 11.18 2.24E−04 2.84E−03 5.27E−03 2.68E−03 2.44E−03 2.66E−03 7.03E−03 3.14E−03 2.35E−03 2.63E−03 11.20 2.51E−04 2.74E−03 5.09E−03 2.61E−03 2.39E−03 2.57E−03 6.80E−03 3.05E−03 2.28E−03 2.55E−03 11.22 2.89E−04 2.65E−03 4.92E−03 2.53E−03 2.30E−03 2.49E−03 6.59E−03 2.96E−03 2.22E−03 2.50E−03 11.23 3.41E−04 2.59E−03 4.76E−03 2.44E−03 2.23E−03 2.42E−03 6.38E−03 2.86E−03 2.15E−03 2.40E−03 11.25 3.80E−04 2.48E−03 4.63E−03 2.37E−03 2.17E−03 2.36E−03 6.22E−03 2.78E−03 2.10E−03 2.33E−03 11.27 4.58E−04 2.41E−03 4.49E−03 2.32E−03 2.11E−03 2.27E−03 6.09E−03 2.69E−03 2.03E−03 2.27E−03 11.28 5.44E−04 2.34E−03 4.37E−03 2.23E−03 2.06E−03 2.22E−03 5.94E−03 2.61E−03 1.98E−03 2.22E−03 11.30 6.34E−04 2.26E−03 4.24E−03 2.17E−03 1.99E−03 2.15E−03 5.79E−03 2.55E−03 1.92E−03 2.14E−03 11.32 7.64E−04 2.20E−03 4.11E−03 2.10E−03 1.94E−03 2.07E−03 5.68E−03 2.47E−03 1.86E−03 2.08E−03 11.33 8.98E−04 2.14E−03 4.02E−03 2.05E−03 1.91E−03 2.02E−03 5.55E−03 2.41E−03 1.81E−03 2.02E−03 11.35 1.04E−03 2.08E−03 3.91E−03 1.99E−03 1.84E−03 1.98E−03 5.41E−03 2.32E−03 1.76E−03 1.98E−03 11.37 1.25E−03 2.02E−03 3.81E−03 1.92E−03 1.78E−03 1.91E−03 5.27E−03 2.26E−03 1.71E−03 1.92E−03 11.38 1.48E−03 1.96E−03 3.70E−03 1.89E−03 1.76E−03 1.88E−03 5.11E−03 2.20E−03 1.67E−03 1.88E−03 11.40 1.76E−03 1.89E−03 3.62E−03 1.82E−03 1.71E−03 1.81E−03 4.96E−03 2.14E−03 1.63E−03 1.83E−03 11.42 2.09E−03 1.84E−03 3.51E−03 1.78E−03 1.67E−03 1.76E−03 4.79E−03 2.08E−03 1.61E−03 1.80E−03 11.43 2.51E−03 1.79E−03 3.43E−03 1.72E−03 1.63E−03 1.72E−03 4.64E−03 2.03E−03 1.55E−03 1.74E−03 11.45 3.05E−03 1.74E−03 3.34E−03 1.68E−03 1.59E−03 1.67E−03 4.46E−03 1.96E−03 1.51E−03 1.69E−03 11.47 3.70E−03 1.70E−03 3.25E−03 1.63E−03 1.55E−03 1.65E−03 4.30E−03 1.90E−03 1.47E−03 1.65E−03 11.48 4.53E−03 1.65E−03 3.16E−03 1.58E−03 1.50E−03 1.59E−03 4.16E−03 1.87E−03 1.43E−03 1.63E−03 11.50 5.54E−03 1.62E−03 3.08E−03 1.55E−03 1.46E−03 1.55E−03 4.00E−03 1.83E−03 1.41E−03 1.57E−03 11.52 6.73E−03 1.59E−03 2.99E−03 1.52E−03 1.44E−03 1.51E−03 3.86E−03 1.77E−03 1.38E−03 1.52E−03 11.53 8.12E−03 1.53E−03 2.92E−03 1.48E−03 1.41E−03 1.48E−03 3.74E−03 1.73E−03 1.33E−03 1.49E−03 11.55 9.72E−03 1.51E−03 2.83E−03 1.45E−03 1.37E−03 1.46E−03 3.61E−03 1.69E−03 1.30E−03 1.47E−03 11.57 1.15E−02 1.50E−03 2.77E−03 1.41E−03 1.35E−03 1.44E−03 3.50E−03 1.64E−03 1.26E−03 1.44E−03 11.58 1.33E−02 1.48E−03 2.71E−03 1.38E−03 1.33E−03 1.43E−03 3.40E−03 1.62E−03 1.25E−03 1.40E−03 11.60 1.52E−02 1.48E−03 2.65E−03 1.37E−03 1.30E−03 1.42E−03 3.33E−03 1.58E−03 1.24E−03 1.38E−03 11.62 1.70E−02 1.49E−03 2.60E−03 1.33E−03 1.28E−03 1.44E−03 3.26E−03 1.56E−03 1.23E−03 1.36E−03 11.63 1.87E−02 1.49E−03 2.60E−03 1.33E−03 1.29E−03 1.44E−03 3.19E−03 1.55E−03 1.19E−03 1.37E−03 11.65 2.03E−02 1.54E−03 2.59E−03 1.32E−03 1.27E−03 1.50E−03 3.15E−03 1.54E−03 1.19E−03 1.36E−03 11.67 2.17E−02 1.60E−03 2.61E−03 1.33E−03 1.27E−03 1.55E−03 3.09E−03 1.53E−03 1.16E−03 1.35E−03 11.68 2.30E−02 1.68E−03 2.64E−03 1.33E−03 1.28E−03 1.60E−03 3.08E−03 1.52E−03 1.15E−03 1.36E−03 11.70 2.44E−02 1.75E−03 2.68E−03 1.32E−03 1.24E−03 1.66E−03 3.01E−03 1.52E−03 1.12E−03 1.36E−03 11.72 2.63E−02 1.80E−03 2.72E−03 1.35E−03 1.24E−03 1.72E−03 2.96E−03 1.50E−03 1.10E−03 1.36E−03 11.73 2.92E−02 1.88E−03 2.76E−03 1.35E−03 1.23E−03 1.77E−03 2.90E−03 1.48E−03 1.09E−03 1.35E−03 11.75 3.40E−02 1.91E−03 2.78E−03 1.36E−03 1.21E−03 1.79E−03 2.82E−03 1.44E−03 1.05E−03 1.35E−03 11.77 4.19E−02 1.92E−03 2.81E−03 1.34E−03 1.20E−03 1.80E−03 2.73E−03 1.43E−03 1.04E−03 1.33E−03 11.78 5.41E−02 1.90E−03 2.79E−03 1.33E−03 1.17E−03 1.79E−03 2.64E−03 1.38E−03 1.01E−03 1.31E−03 11.80 7.29E−02 1.87E−03 2.73E−03 1.31E−03 1.13E−03 1.74E−03 2.51E−03 1.33E−03 9.67E−04 1.26E−03 11.82 9.97E−02 1.80E−03 2.66E−03 1.26E−03 1.09E−03 1.68E−03 2.42E−03 1.29E−03 9.61E−04 1.23E−03 11.83 1.36E−01 1.71E−03 2.57E−03 1.24E−03 1.05E−03 1.61E−03 2.31E−03 1.23E−03 9.10E−04 1.18E−03 11.85 1.81E−01 1.61E−03 2.43E−03 1.17E−03 1.03E−03 1.51E−03 2.21E−03 1.17E−03 8.75E−04 1.14E−03 11.87 2.34E−01 1.48E−03 2.31E−03 1.13E−03 9.87E−04 1.42E−03 2.12E−03 1.13E−03 8.54E−04 1.09E−03 11.88 2.94E−01 1.37E−03 2.18E−03 1.07E−03 9.50E−04 1.32E−03 2.03E−03 1.08E−03 8.39E−04 1.04E−03 11.90 3.55E−01 1.28E−03 2.03E−03 1.03E−03 8.93E−04 1.22E−03 1.95E−03 1.05E−03 8.43E−04 1.01E−03 11.92 4.11E−01 1.18E−03 1.91E−03 9.81E−04 8.73E−04 1.15E−03 1.87E−03 1.01E−03 7.94E−04 9.87E−04 11.93 4.57E−01 1.09E−03 1.80E−03 9.33E−04 8.72E−04 1.09E−03 1.83E−03 9.66E−04 7.86E−04 9.47E−04 11.95 4.88E−01 1.02E−03 1.70E−03 8.88E−04 8.44E−04 1.03E−03 1.74E−03 9.40E−04 7.79E−04 9.16E−04 11.97 5.02E−01 9.48E−04 1.62E−03 8.68E−04 8.29E−04 9.85E−04 1.69E−03 9.11E−04 7.62E−04 8.29E−04 11.98 4.96E−01 8.99E−04 1.54E−03 8.37E−04 8.11E−04 9.34E−04 1.65E−03 8.93E−04 7.50E−04 8.88E−04 12.00 4.73E−01 8.71E−04 1.46E−03 8.22E−04 8.00E−04 9.07E−04 1.60E−03 8.77E−04 7.27E−04 8.65E−04 12.02 4.37E−01 8.32E−04 1.40E−03 7.96E−04 7.76E−04 8.98E−04 1.54E−03 8.30E−04 7.12E−04 8.43E−04 12.03 3.91E−01 7.88E−04 1.34E−03 7.82E−04 7.71E−04 8.54E−04 1.51E−03 8.18E−04 7.04E−04 8.30E−04 12.05 3.40E−01 7.63E−04 1.31E−03 7.70E−04 7.47E−04 8.29E−04 1.47E−03 8.06E−04 6.91E−04 8.23E−04 12.07 2.89E−01 7.49E−04 1.27E−03 7.45E−04 7.42E−04 8.25E−04 1.43E−03 7.78E−04 6.77E−04 8.08E−04 12.08 2.42E−01 7.05E−04 1.22E−03 7.42E−04 7.17E−04 8.32E−04 1.37E−03 7.73E−04 6.63E−04 7.96E−04 12.10 2.00E−01 7.00E−04 1.18E−03 7.32E−04 6.98E−04 7.89E−04 1.35E−03 7.50E−04 6.42E−04 7.55E−04 12.12 1.64E−01 6.83E−04 1.16E−03 7.12E−04 6.98E−04 7.74E−04 1.31E−03 7.51E−04 6.43E−04 7.61E−04 12.13 1.33E−01 6.62E−04 1.14E−03 7.00E−04 6.78E−04 7.45E−04 1.28E−03 7.22E−04 6.29E−04 7.47E−04 12.15 1.09E−01 6.55E−04 1.12E−03 6.73E−04 6.58E−04 7.26E−04 1.23E−03 7.14E−04 6.08E−04 7.29E−04 12.17 9.03E−02 6.27E−04 1.07E−03 6.50E−04 6.44E−04 7.09E−04 1.21E−03 6.84E−04 6.03E−04 7.07E−04 12.18 7.53E−02 6.11E−04 1.05E−03 6.42E−04 6.40E−04 6.85E−04 1.17E−03 6.77E−04 5.84E−04 6.95E−04 12.20 6.33E−02 5.86E−04 1.02E−03 6.19E−04 6.26E−04 6.74E−04 1.14E−03 6.70E−04 5.89E−04 6.92E−04 12.22 5.38E−02 5.96E−04 1.01E−03 6.09E−04 6.14E−04 6.47E−04 1.12E−03 6.42E−04 5.82E−04 6.76E−04 12.23 4.63E−02 5.75E−04 9.96E−04 5.95E−04 6.05E−04 6.54E−04 1.09E−03 6.42E−04 5.58E−04 6.48E−04 12.25 4.03E−02 5.82E−04 9.61E−04 5.84E−04 5.97E−04 6.26E−04 1.07E−03 6.28E−04 5.38E−04 6.46E−04 12.27 3.54E−02 5.48E−04 9.48E−04 5.66E−04 5.96E−04 6.17E−04 1.04E−03 6.19E−04 5.48E−04 6.29E−04 12.28 3.12E−02 5.46E−04 9.21E−04 5.86E−04 5.69E−04 6.06E−04 1.03E−03 6.23E−04 5.33E−04 6.36E−04 12.30 2.79E−02 5.29E−04 9.29E−04 5.48E−04 5.78E−04 5.94E−04 1.00E−03 5.97E−04 5.47E−04 6.26E−04 12.32 2.50E−02 5.26E−04 8.78E−04 5.38E−04 5.73E−04 5.82E−04 9.77E−04 5.94E−04 5.37E−04 6.26E−04 12.33 2.26E−02 5.14E−04 8.58E−04 5.26E−04 5.63E−04 5.81E−04 9.56E−04 5.86E−04 5.30E−04 6.15E−04 12.35 2.05E−02 5.06E−04 8.44E−04 5.37E−04 5.52E−04 5.64E−04 9.34E−04 5.58E−04 5.05E−04 6.13E−04 12.37 1.87E−02 5.01E−04 8.27E−04 5.28E−04 5.55E−04 5.53E−04 9.18E−04 5.63E−04 5.07E−04 5.98E−04 12.38 1.71E−02 4.78E−04 8.21E−04 5.14E−04 5.49E−04 5.69E−04 9.09E−04 5.47E−04 5.00E−04 6.04E−04 12.40 1.58E−02 4.73E−04 7.93E−04 5.35E−04 5.38E−04 5.74E−04 8.75E−04 5.63E−04 5.13E−04 5.86E−04 12.42 1.47E−02 4.56E−04 7.81E−04 5.09E−04 5.53E−04 5.55E−04 8.63E−04 5.55E−04 4.86E−04 5.87E−04 12.43 1.36E−02 4.58E−04 7.59E−04 5.06E−04 5.28E−04 5.53E−04 8.29E−04 5.45E−04 4.89E−04 5.81E−04 12.45 1.27E−02 4.46E−04 7.54E−04 5.09E−04 5.34E−04 5.48E−04 8.25E−04 5.14E−04 4.88E−04 5.77E−04 12.47 1.20E−02 4.35E−04 7.31E−04 5.04E−04 5.20E−04 5.53E−04 7.98E−04 5.06E−04 4.89E−04 5.66E−04 12.48 1.13E−02 4.42E−04 7.31E−04 4.98E−04 5.26E−04 5.38E−04 7.99E−04 5.13E−04 4.75E−04 5.60E−04 12.50 1.08E−02 4.47E−04 7.19E−04 4.90E−04 4.95E−04 5.13E−04 7.84E−04 4.98E−04 4.57E−04 5.55E−04 12.52 1.02E−02 4.24E−04 7.13E−04 4.84E−04 5.14E−04 5.22E−04 7.64E−04 4.81E−04 4.56E−04 5.47E−04 12.53 9.83E−03 4.32E−04 7.04E−04 5.04E−04 4.96E−04 4.97E−04 7.49E−04 4.88E−04 4.70E−04 5.37E−04 12.55 9.51E−03 4.31E−04 6.85E−04 4.79E−04 4.81E−04 4.98E−04 7.54E−04 4.83E−04 4.75E−04 5.12E−04 12.57 9.23E−03 4.07E−04 6.95E−04 4.56E−04 4.75E−04 4.79E−04 7.30E−04 4.74E−04 4.51E−04 5.19E−04 12.58 9.00E−03 4.10E−04 6.95E−04 4.54E−04 4.61E−04 4.69E−04 7.34E−04 4.89E−04 4.46E−04 5.18E−04 12.60 8.86E−03 4.09E−04 6.94E−04 4.55E−04 4.71E−04 4.71E−04 7.35E−04 4.81E−04 4.58E−04 5.16E−04 12.62 8.75E−03 4.18E−04 6.96E−04 4.65E−04 4.76E−04 4.71E−04 7.30E−04 4.70E−04 4.50E−04 5.07E−04 12.63 8.66E−03 4.32E−04 6.73E−04 4.50E−04 5.05E−04 4.81E−04 7.42E−04 4.75E−04 4.67E−04 5.14E−04 12.65 8.57E−03 4.30E−04 6.80E−04 4.90E−04 5.35E−04 4.99E−04 7.52E−04 4.68E−04 4.79E−04 5.15E−04 12.67 8.45E−03 4.48E−04 6.86E−04 4.95E−04 5.74E−04 5.08E−04 7.45E−04 4.84E−04 4.81E−04 5.24E−04 12.68 8.32E−03 4.75E−04 6.66E−04 5.36E−04 6.29E−04 5.30E−04 7.75E−04 4.81E−04 4.96E−04 5.19E−04 12.70 8.18E−03 4.90E−04 6.60E−04 6.02E−04 6.98E−04 5.95E−04 8.27E−04 5.07E−04 5.30E−04 5.27E−04 12.72 7.99E−03 5.04E−04 6.47E−04 6.74E−04 8.18E−04 6.28E−04 8.83E−04 5.39E−04 5.69E−04 5.24E−04 12.73 7.81E−03 5.40E−04 6.51E−04 7.56E−04 9.43E−04 7.43E−04 9.81E−04 5.86E−04 6.21E−04 5.48E−04 12.75 7.62E−03 5.92E−04 6.46E−04 8.87E−04 1.13E−03 8.59E−04 1.09E−03 6.54E−04 7.01E−04 5.59E−04 12.77 7.41E−03 6.31E−04 6.62E−04 1.08E−03 1.36E−03 1.01E−03 1.18E−03 7.36E−04 7.65E−04 5.84E−04 12.78 7.25E−03 7.09E−04 6.93E−04 1.31E−03 1.58E−03 1.21E−03 1.26E−03 8.28E−04 8.28E−04 6.17E−04 12.80 7.09E−03 8.28E−04 7.25E−04 1.60E−03 1.78E−03 1.42E−03 1.32E−03 9.03E−04 8.75E−04 6.71E−04 12.82 6.92E−03 9.60E−04 7.52E−04 1.86E−03 1.88E−03 1.57E−03 1.32E−03 9.68E−04 8.98E−04 7.22E−04 12.83 6.74E−03 1.11E−03 7.75E−04 2.09E−03 1.91E−03 1.67E−03 1.31E−03 1.00E−03 9.29E−04 7.72E−04 12.85 6.57E−03 1.29E−03 8.22E−04 2.21E−03 1.81E−03 1.67E−03 1.27E−03 1.01E−03 9.01E−04 8.45E−04 12.87 6.41E−03 1.44E−03 8.53E−04 2.21E−03 1.68E−03 1.62E−03 1.22E−03 1.01E−03 8.54E−04 8.67E−04 12.88 6.25E−03 1.60E−03 8.67E−04 2.08E−03 1.50E−03 1.48E−03 1.13E−03 9.89E−04 8.11E−04 8.84E−04 12.90 6.08E−03 1.68E−03 8.64E−04 1.89E−03 1.29E−03 1.31E−03 1.05E−03 9.43E−04 7.65E−04 8.94E−04 12.92 5.89E−03 1.72E−03 8.62E−04 1.64E−03 1.14E−03 1.14E−03 9.62E−04 8.68E−04 7.10E−04 8.51E−04 12.93 5.72E−03 1.66E−03 8.22E−04 1.40E−03 9.86E−04 9.82E−04 8.67E−04 7.85E−04 6.31E−04 8.13E−04 12.95 5.56E−03 1.55E−03 7.74E−04 1.18E−03 8.51E−04 8.54E−04 8.00E−04 7.16E−04 5.95E−04 7.69E−04 12.97 5.41E−03 1.39E−03 7.31E−04 1.00E−03 7.71E−04 7.56E−04 7.19E−04 6.35E−04 5.44E−04 7.11E−04 12.98 5.21E−03 1.21E−03 6.86E−04 8.58E−04 6.95E−04 6.82E−04 6.71E−04 5.96E−04 5.19E−04 6.46E−04 13.00 5.06E−03 1.04E−03 6.38E−04 7.49E−04 6.29E−04 6.28E−04 6.07E−04 5.18E−04 4.79E−04 5.98E−04 13.02 4.94E−03 8.82E−04 5.93E−04 6.71E−04 5.79E−04 5.77E−04 5.77E−04 4.89E−04 4.50E−04 5.70E−04 13.03 4.81E−03 7.54E−04 5.60E−04 6.28E−04 5.78E−04 5.42E−04 5.47E−04 4.44E−04 4.42E−04 5.44E−04 13.05 4.67E−03 6.65E−04 5.32E−04 5.76E−04 5.35E−04 5.22E−04 5.25E−04 4.00E−04 4.20E−04 4.90E−04 13.07 4.52E−03 5.75E−04 5.12E−04 5.58E−04 5.06E−04 4.90E−04 5.08E−04 3.95E−04 3.98E−04 4.84E−04 13.08 4.41E−03 5.03E−04 5.00E−04 5.13E−04 5.06E−04 4.78E−04 4.88E−04 3.77E−04 3.75E−04 4.85E−04 13.10 4.30E−03 4.68E−04 4.68E−04 4.88E−04 4.91E−04 4.63E−04 4.80E−04 3.68E−04 3.57E−04 4.59E−04 13.12 4.21E−03 4.36E−04 4.67E−04 4.58E−04 4.54E−04 4.40E−04 4.42E−04 3.42E−04 3.50E−04 4.45E−04 13.13 4.14E−03 4.05E−04 4.40E−04 4.49E−04 4.60E−04 4.38E−04 4.30E−04 3.42E−04 3.48E−04 4.36E−04 13.15 3.99E−03 3.86E−04 4.32E−04 4.30E−04 4.41E−04 4.01E−04 4.20E−04 3.14E−04 3.36E−04 4.32E−04 13.17 3.91E−03 3.55E−04 4.28E−04 4.26E−04 4.29E−04 3.96E−04 4.18E−04 3.13E−04 3.36E−04 3.98E−04 13.18 3.83E−03 3.38E−04 4.15E−04 4.02E−04 4.27E−04 3.91E−04 4.00E−04 2.91E−04 3.20E−04 4.16E−04 13.20 3.76E−03 3.40E−04 4.25E−04 3.97E−04 4.28E−04 3.89E−04 4.06E−04 2.90E−04 3.22E−04 4.07E−04 13.22 3.66E−03 3.19E−04 4.10E−04 3.89E−04 4.21E−04 3.97E−04 3.88E−04 2.91E−04 3.11E−04 4.05E−04 13.23 3.57E−03 3.04E−04 4.17E−04 3.72E−04 4.22E−04 3.68E−04 3.86E−04 2.86E−04 3.09E−04 3.96E−04 13.25 3.47E−03 3.00E−04 3.98E−04 3.87E−04 4.10E−04 3.72E−04 3.73E−04 2.73E−04 3.12E−04 3.96E−04 13.27 3.41E−03 2.92E−04 3.88E−04 3.55E−04 4.11E−04 3.68E−04 3.77E−04 2.63E−04 2.92E−04 3.89E−04 13.28 3.34E−03 2.93E−04 3.75E−04 3.55E−04 4.15E−04 3.59E−04 3.58E−04 2.69E−04 3.06E−04 4.01E−04 13.30 3.24E−03 3.00E−04 3.72E−04 3.67E−04 3.94E−04 3.55E−04 3.70E−04 2.66E−04 3.02E−04 3.97E−04 13.32 3.21E−03 2.76E−04 3.68E−04 3.65E−04 3.95E−04 3.67E−04 3.51E−04 2.54E−04 2.84E−04 3.92E−04 13.33 3.11E−03 2.67E−04 3.78E−04 3.47E−04 3.72E−04 3.46E−04 3.47E−04 2.48E−04 3.03E−04 3.98E−04 13.35 3.04E−03 2.67E−04 3.63E−04 3.55E−04 3.76E−04 3.29E−04 3.36E−04 2.45E−04 2.90E−04 3.73E−04 13.37 2.97E−03 2.68E−04 3.61E−04 3.20E−04 3.58E−04 3.21E−04 3.32E−04 2.43E−04 2.76E−04 3.82E−04 13.38 2.88E−03 2.77E−04 3.50E−04 3.30E−04 3.49E−04 3.70E−04 3.13E−04 2.21E−04 2.69E−04 3.71E−04 13.40 2.80E−03 2.62E−04 3.42E−04 3.11E−04 3.52E−04 3.26E−04 3.22E−04 2.20E−04 2.77E−04 3.77E−04 13.42 2.73E−03 2.52E−04 3.53E−04 2.99E−04 3.31E−04 3.08E−04 3.17E−04 2.18E−04 2.78E−04 3.66E−04 13.43 2.68E−03 2.39E−04 3.38E−04 3.10E−04 3.47E−04 3.12E−04 3.03E−04 2.28E−04 2.77E−04 3.63E−04 13.45 2.59E−03 2.41E−04 3.36E−04 2.84E−04 3.38E−04 2.92E−04 3.08E−04 2.14E−04 2.56E−04 3.65E−04 13.47 2.50E−03 2.47E−04 3.35E−04 2.88E−04 3.19E−04 3.08E−04 3.03E−04 2.14E−04 2.58E−04 3.65E−04 13.48 2.43E−03 2.47E−04 3.25E−04 2.98E−04 3.27E−04 2.94E−04 3.03E−04 2.00E−04 2.59E−04 3.70E−04 13.50 2.37E−03 2.42E−04 3.03E−04 2.82E−04 3.18E−04 2.94E−04 2.97E−04 2.01E−04 2.63E−04 3.49E−04 13.52 2.29E−03 2.47E−04 2.96E−04 2.81E−04 3.29E−04 2.82E−04 3.03E−04 2.01E−04 2.58E−04 3.58E−04 13.53 2.23E−03 2.45E−04 3.01E−04 2.71E−04 3.18E−04 2.79E−04 2.82E−04 1.92E−04 2.62E−04 3.56E−04 13.55 2.14E−03 2.30E−04 3.03E−04 2.86E−04 3.28E−04 2.78E−04 2.76E−04 1.89E−04 2.45E−04 3.52E−04 13.57 2.07E−03 2.25E−04 3.11E−04 2.70E−04 2.96E−04 2.87E−04 2.87E−04 1.75E−04 2.45E−04 3.59E−04 13.58 2.01E−03 2.37E−04 3.03E−04 2.69E−04 3.17E−04 2.79E−04 2.66E−04 1.81E−04 2.54E−04 3.42E−04 13.60 1.94E−03 2.27E−04 3.00E−04 2.81E−04 3.16E−04 2.82E−04 2.63E−04 1.82E−04 2.56E−04 3.50E−04 13.62 1.86E−03 2.14E−04 3.10E−04 2.76E−04 2.92E−04 2.86E−04 2.87E−04 1.68E−04 2.53E−04 3.40E−04 13.63 1.80E−03 2.25E−04 2.77E−04 2.63E−04 2.89E−04 2.71E−04 2.49E−04 1.91E−04 2.57E−04 3.53E−04 13.65 1.75E−03 2.21E−04 2.87E−04 2.68E−04 2.98E−04 2.83E−04 2.59E−04 1.71E−04 2.57E−04 3.27E−04 13.67 1.68E−03 2.29E−04 2.80E−04 2.43E−04 3.02E−04 2.74E−04 2.53E−04 1.71E−04 2.44E−04 3.26E−04 13.68 1.65E−03 2.37E−04 2.83E−04 2.50E−04 2.98E−04 2.79E−04 2.41E−04 1.53E−04 2.57E−04 3.32E−04 13.70 1.60E−03 2.17E−04 2.73E−04 2.41E−04 2.91E−04 2.60E−04 2.35E−04 1.62E−04 2.43E−04 3.31E−04 13.72 1.54E−03 2.07E−04 2.60E−04 2.52E−04 2.82E−04 2.66E−04 2.37E−04 1.65E−04 2.31E−04 3.25E−04 13.73 1.47E−03 2.03E−04 2.67E−04 2.43E−04 2.91E−04 2.82E−04 2.32E−04 1.62E−04 2.58E−04 3.26E−04 13.75 1.43E−03 2.12E−04 2.64E−04 2.43E−04 2.91E−04 2.58E−04 2.27E−04 1.66E−04 2.37E−04 3.22E−04 13.77 1.39E−03 2.05E−04 2.59E−04 2.30E−04 2.81E−04 2.82E−04 2.20E−04 1.62E−04 2.41E−04 3.26E−04 13.78 1.33E−03 2.18E−04 2.63E−04 2.25E−04 2.73E−04 2.50E−04 2.17E−04 1.46E−04 2.32E−04 3.12E−04 13.80 1.30E−03 2.13E−04 2.49E−04 2.30E−04 2.82E−04 2.59E−04 2.28E−04 1.59E−04 2.42E−04 3.27E−04 13.82 1.25E−03 2.09E−04 2.42E−04 2.35E−04 2.72E−04 2.54E−04 2.24E−04 1.60E−04 2.32E−04 3.13E−04 13.83 1.25E−03 2.11E−04 2.50E−04 2.34E−04 2.70E−04 2.66E−04 2.05E−04 1.42E−04 2.34E−04 3.11E−04 13.85 1.20E−03 1.98E−04 2.44E−04 2.24E−04 2.53E−04 2.59E−04 2.02E−04 1.40E−04 2.30E−04 3.20E−04 13.87 1.14E−03 2.03E−04 2.34E−04 2.25E−04 2.61E−04 2.61E−04 2.07E−04 1.52E−04 2.29E−04 3.31E−04 13.88 1.10E−03 1.89E−04 2.30E−04 2.22E−04 2.72E−04 2.63E−04 2.01E−04 1.45E−04 2.30E−04 3.10E−04 13.90 1.07E−03 1.97E−04 2.39E−04 2.11E−04 2.61E−04 2.49E−04 2.03E−04 1.45E−04 2.28E−04 3.02E−04 13.92 1.04E−03 1.99E−04 2.23E−04 2.09E−04 2.51E−04 2.45E−04 1.86E−04 1.46E−04 2.25E−04 3.22E−04 13.93 1.02E−03 2.00E−04 2.32E−04 2.19E−04 2.58E−04 2.38E−04 1.94E−04 1.49E−04 2.28E−04 3.02E−04 13.95 9.71E−04 1.90E−04 2.22E−04 2.13E−04 2.52E−04 2.52E−04 1.90E−04 1.38E−04 2.31E−04 3.20E−04 13.97 9.52E−04 1.80E−04 2.20E−04 2.02E−04 2.60E−04 2.50E−04 2.05E−04 1.39E−04 2.37E−04 3.03E−04 13.98 9.27E−04 1.89E−04 2.25E−04 2.20E−04 2.61E−04 2.44E−04 1.83E−04 1.38E−04 2.32E−04 3.01E−04 14.00 9.02E−04 1.80E−04 2.23E−04 1.92E−04 2.71E−04 2.34E−04 1.83E−04 1.36E−04 2.29E−04 2.99E−04 14.02 8.91E−04 1.70E−04 2.20E−04 1.95E−04 2.45E−04 2.47E−04 1.80E−04 1.36E−04 2.35E−04 3.09E−04 14.03 8.64E−04 1.90E−04 2.18E−04 2.01E−04 2.51E−04 2.29E−04 1.75E−04 1.19E−04 2.27E−04 2.96E−04 14.05 8.38E−04 1.93E−04 2.19E−04 2.05E−04 2.31E−04 2.42E−04 1.86E−04 1.36E−04 2.35E−04 2.99E−04 14.07 8.18E−04 1.73E−04 2.23E−04 2.10E−04 2.42E−04 2.35E−04 1.68E−04 1.25E−04 2.22E−04 3.00E−04 14.08 7.92E−04 1.81E−04 2.05E−04 2.01E−04 2.14E−04 2.24E−04 1.65E−04 1.21E−04 2.24E−04 3.00E−04 14.10 7.82E−04 1.69E−04 2.07E−04 1.94E−04 2.25E−04 2.44E−04 1.69E−04 1.32E−04 2.35E−04 3.02E−04 14.12 7.61E−04 1.86E−04 2.02E−04 1.90E−04 2.37E−04 2.47E−04 1.81E−04 1.26E−04 2.49E−04 2.93E−04 14.13 7.49E−04 1.78E−04 2.22E−04 1.86E−04 2.35E−04 2.23E−04 1.81E−04 1.15E−04 2.23E−04 2.93E−04 14.15 7.32E−04 1.73E−04 2.03E−04 2.00E−04 2.42E−04 2.42E−04 1.83E−04 1.23E−04 2.21E−04 2.97E−04 14.17 7.13E−04 1.66E−04 2.00E−04 1.82E−04 2.28E−04 2.28E−04 1.69E−04 1.15E−04 2.21E−04 2.90E−04 14.18 7.03E−04 1.66E−04 2.07E−04 1.93E−04 2.39E−04 2.35E−04 1.66E−04 1.16E−04 2.37E−04 2.83E−04 14.20 6.75E−04 1.68E−04 2.05E−04 1.90E−04 2.32E−04 2.32E−04 1.55E−04 1.49E−04 2.40E−04 2.89E−04 14.22 6.70E−04 1.70E−04 2.05E−04 1.92E−04 2.33E−04 2.29E−04 1.69E−04 1.20E−04 2.17E−04 2.86E−04 14.23 6.54E−04 1.81E−04 1.86E−04 1.89E−04 2.23E−04 2.29E−04 1.69E−04 1.16E−04 2.27E−04 2.82E−04 14.25 6.41E−04 1.95E−04 2.12E−04 1.84E−04 2.40E−04 2.29E−04 1.65E−04 1.35E−04 2.22E−04 2.89E−04 14.27 6.28E−04 1.68E−04 1.91E−04 1.83E−04 2.22E−04 2.30E−04 1.55E−04 1.11E−04 2.11E−04 2.81E−04 14.28 6.40E−04 1.75E−04 1.90E−04 1.90E−04 2.25E−04 2.42E−04 1.69E−04 1.07E−04 2.35E−04 2.83E−04 14.30 6.06E−04 1.72E−04 1.84E−04 1.84E−04 2.22E−04 2.37E−04 1.55E−04 1.25E−04 2.21E−04 2.84E−04 14.32 5.98E−04 1.81E−04 1.89E−04 1.76E−04 2.28E−04 2.15E−04 1.79E−04 1.07E−04 2.32E−04 2.98E−04 14.33 5.91E−04 1.82E−04 1.88E−04 1.82E−04 2.24E−04 2.19E−04 1.56E−04 1.05E−04 2.24E−04 2.81E−04 14.35 5.75E−04 1.80E−04 2.00E−04 1.73E−04 2.32E−04 2.25E−04 1.52E−04 1.11E−04 2.27E−04 2.92E−04 14.37 5.62E−04 1.58E−04 1.93E−04 1.79E−04 2.24E−04 2.21E−04 1.66E−04 1.12E−04 2.20E−04 2.69E−04 14.38 5.57E−04 1.76E−04 2.02E−04 1.81E−04 2.25E−04 2.34E−04 1.61E−04 1.10E−04 2.25E−04 2.86E−04 14.40 5.42E−04 1.69E−04 1.83E−04 1.82E−04 2.20E−04 2.24E−04 1.49E−04 1.15E−04 2.29E−04 2.91E−04 14.42 5.25E−04 1.88E−04 1.90E−04 1.78E−04 2.37E−04 2.20E−04 1.53E−04 1.09E−04 2.21E−04 2.70E−04 14.43 5.26E−04 1.69E−04 1.95E−04 1.71E−04 2.10E−04 2.08E−04 1.59E−04 9.30E−05 2.20E−04 2.71E−04 14.45 5.26E−04 1.64E−04 1.97E−04 1.80E−04 2.08E−04 2.19E−04 1.53E−04 1.03E−04 2.14E−04 2.73E−04 14.47 5.19E−04 1.74E−04 2.00E−04 1.84E−04 2.13E−04 2.08E−04 1.71E−04 1.11E−04 2.35E−04 2.64E−04 14.48 5.06E−04 1.76E−04 1.78E−04 1.75E−04 2.22E−04 2.18E−04 1.58E−04 1.13E−04 2.23E−04 2.74E−04 14.50 4.93E−04 1.68E−04 1.88E−04 1.59E−04 2.02E−04 2.17E−04 1.62E−04 9.90E−04 2.31E−04 2.77E−04 14.52 4.95E−04 1.63E−04 1.98E−04 1.72E−04 2.11E−04 2.05E−04 1.60E−04 1.05E−04 2.30E−04 3.02E−04 14.53 4.78E−04 1.60E−04 2.00E−04 1.69E−04 1.98E−04 2.04E−04 1.49E−04 1.01E−04 2.17E−04 2.79E−04 14.55 4.78E−04 1.80E−04 1.99E−04 1.98E−04 2.15E−04 2.17E−04 1.69E−04 1.03E−04 2.24E−04 2.74E−04 14.57 4.81E−04 1.73E−04 1.78E−04 1.59E−04 2.13E−04 1.93E−04 1.56E−04 1.00E−04 2.25E−04 2.63E−04 14.58 4.65E−04 1.52E−04 1.86E−04 1.72E−04 1.97E−04 2.09E−04 1.58E−04 9.60E−05 2.33E−04 2.73E−04 14.60 4.83E−04 1.58E−04 1.84E−04 1.61E−04 2.12E−04 2.03E−04 1.65E−04 9.60E−05 2.32E−04 2.66E−04 14.62 4.46E−04 1.48E−04 1.92E−04 1.53E−04 2.02E−04 2.10E−04 1.55E−04 1.17E−04 2.29E−04 2.73E−04 14.63 4.54E−04 1.72E−04 1.89E−04 1.63E−04 2.07E−04 2.01E−04 1.62E−04 1.03E−04 2.38E−04 2.63E−04 14.65 4.41E−04 1.83E−04 1.85E−04 1.42E−04 1.99E−04 2.03E−04 1.68E−04 9.90E−05 2.39E−04 2.69E−04 14.67 4.37E−04 1.65E−04 1.91E−04 1.66E−04 2.19E−04 2.05E−04 1.79E−04 1.12E−04 2.51E−04 2.82E−04 14.68 4.35E−04 1.54E−04 1.95E−04 1.60E−04 2.03E−04 1.99E−04 1.66E−04 1.02E−04 2.68E−04 2.68E−04 14.70 4.22E−04 1.72E−04 1.86E−04 1.53E−04 2.10E−04 1.91E−04 1.73E−04 1.03E−04 2.60E−04 2.73E−04 14.72 4.18E−04 1.66E−04 2.10E−04 1.51E−04 2.21E−04 2.12E−04 1.80E−04 1.07E−04 2.53E−04 2.70E−04 14.73 4.24E−04 1.64E−04 1.98E−04 1.59E−04 2.17E−04 2.04E−04 1.81E−04 1.21E−04 2.71E−04 2.79E−04 14.75 4.18E−04 1.68E−04 2.04E−04 1.59E−04 2.20E−04 2.11E−04 1.85E−04 1.23E−04 2.61E−04 2.84E−04 14.77 4.54E−04 1.63E−04 1.89E−04 1.42E−04 2.39E−04 2.12E−04 1.81E−04 1.23E−04 2.67E−04 2.86E−04 14.78 4.14E−04 1.79E−04 2.04E−04 1.62E−04 2.15E−04 2.11E−04 1.75E−04 1.28E−04 2.62E−04 2.80E−04 14.80 3.79E−04 1.72E−04 1.90E−04 1.60E−04 2.24E−04 2.10E−04 1.93E−04 1.12E−04 2.66E−04 2.77E−04 14.82 3.91E−04 1.82E−04 1.95E−04 1.68E−04 2.30E−04 2.09E−04 1.75E−04 1.15E−04 2.60E−04 2.87E−04 14.83 3.87E−04 1.74E−04 2.02E−04 1.65E−04 2.17E−04 2.05E−04 1.78E−04 1.11E−04 2.30E−04 2.94E−04 14.85 3.76E−04 1.76E−04 2.12E−04 1.65E−04 2.03E−04 2.09E−04 1.75E−04 1.12E−04 2.43E−04 2.86E−04 14.87 3.68E−04 1.75E−04 2.01E−04 1.53E−04 2.12E−04 2.07E−04 1.84E−04 9.90E−05 2.38E−04 2.64E−04 14.88 3.77E−04 1.63E−04 1.92E−04 1.70E−04 2.21E−04 1.98E−04 1.65E−04 1.05E−04 2.45E−04 2.66E−04 14.90 3.65E−04 1.52E−04 1.85E−04 1.51E−04 1.97E−04 1.99E−04 1.85E−04 1.09E−04 2.34E−04 2.61E−04 14.92 3.69E−04 1.63E−04 1.99E−04 1.56E−04 1.90E−04 1.82E−04 1.89E−04 1.01E−04 2.42E−04 2.60E−04 14.93 3.56E−04 1.65E−04 1.80E−04 1.52E−04 1.97E−04 2.03E−04 1.66E−04 9.50E−05 2.25E−04 2.57E−04 14.95 3.46E−04 1.58E−04 1.85E−04 1.41E−04 2.07E−04 2.00E−04 1.83E−04 9.10E−05 2.48E−04 2.53E−04 14.97 3.39E−04 1.54E−04 1.76E−04 1.69E−04 2.00E−04 1.85E−04 1.90E−04 8.20E−05 2.29E−04 2.50E−04 14.98 3.47E−04 1.44E−04 1.84E−04 1.29E−04 1.93E−04 1.91E−04 1.86E−04 9.70E−05 2.42E−04 2.54E−04 15.00 3.52E−04 1.41E−04 1.82E−04 1.38E−04 1.91E−04 1.86E−04 1.82E−04 1.02E−04 2.30E−04 2.43E−04 15.02 3.30E−04 1.51E−04 1.81E−04 1.39E−04 1.97E−04 2.01E−04 1.76E−04 1.02E−04 2.32E−04 2.51E−04 15.03 3.35E−04 1.53E−04 1.90E−04 1.45E−04 1.92E−04 1.86E−04 1.71E−04 8.50E−05 2.32E−04 2.57E−04 15.05 3.42E−04 1.40E−04 1.73E−04 1.46E−04 1.93E−04 1.91E−04 1.81E−04 7.60E−05 2.21E−04 2.56E−04 15.07 3.18E−04 1.34E−04 1.76E−04 1.36E−04 2.02E−04 1.89E−04 1.64E−04 9.10E−05 2.35E−04 2.62E−04 15.08 3.30E−04 1.40E−04 1.83E−04 1.30E−04 1.94E−04 1.83E−04 1.85E−04 9.30E−05 2.37E−04 2.38E−04 15.10 3.27E−04 1.31E−04 1.76E−04 1.35E−04 1.85E−04 1.79E−04 1.74E−04 9.00E−05 2.15E−04 2.60E−04 15.12 3.12E−04 1.32E−04 1.81E−04 1.40E−04 1.88E−04 1.92E−04 1.88E−04 8.10E−05 2.35E−04 2.49E−04 15.13 3.14E−04 1.44E−04 1.89E−04 1.43E−04 1.89E−04 1.85E−04 1.65E−04 8.60E−05 2.23E−04 2.51E−04 15.15 3.13E−04 1.19E−04 1.85E−04 1.51E−04 1.85E−04 1.95E−04 1.74E−04 7.60E−05 2.28E−04 2.31E−04 15.17 3.02E−04 1.25E−04 1.85E−04 1.34E−04 1.78E−04 1.83E−04 1.79E−04 1.02E−04 2.39E−04 2.34E−04 15.18 3.12E−04 1.39E−04 1.83E−04 1.28E−04 1.78E−04 1.73E−04 2.04E−04 8.70E−05 2.35E−04 2.39E−04 15.20 3.12E−04 1.22E−04 1.61E−04 1.24E−04 1.70E−04 1.85E−04 1.75E−04 8.40E−05 2.25E−04 2.57E−04 15.22 3.08E−04 1.23E−04 1.69E−04 1.29E−04 1.70E−04 1.65E−04 1.91E−04 8.10E−05 2.25E−04 2.35E−04 15.23 3.11E−04 1.29E−04 1.80E−04 1.39E−04 1.80E−04 1.74E−04 1.73E−04 9.10E−05 2.23E−04 2.37E−04 15.25 3.00E−04 1.26E−04 1.78E−04 1.35E−04 1.70E−04 1.73E−04 1.73E−04 7.40E−05 2.23E−04 2.40E−04 15.27 2.98E−04 1.14E−04 1.86E−04 1.29E−04 1.75E−04 1.84E−04 1.74E−04 9.10E−05 2.20E−04 2.69E−04 15.28 2.89E−04 1.11E−04 1.62E−04 1.09E−04 1.76E−04 1.88E−04 1.85E−04 7.60E−05 2.13E−04 2.25E−04 15.30 2.94E−04 1.21E−04 1.65E−04 1.16E−04 1.69E−04 1.79E−04 1.66E−04 8.10E−05 2.11E−04 2.41E−04 15.32 2.96E−04 1.07E−04 1.71E−04 1.12E−04 1.74E−04 1.79E−04 1.72E−04 8.20E−05 2.20E−04 2.47E−04 15.33 2.92E−04 1.24E−04 1.62E−04 1.17E−04 1.59E−04 1.70E−04 1.82E−04 6.10E−05 2.03E−04 2.23E−04 15.35 2.99E−04 1.16E−04 1.66E−04 1.16E−04 1.68E−04 1.62E−04 1.73E−04 7.40E−05 2.24E−04 2.47E−04 15.37 3.07E−04 1.19E−04 1.76E−04 1.06E−04 1.72E−04 1.69E−04 1.76E−04 7.00E−05 2.14E−04 2.39E−04 15.38 2.99E−04 1.14E−04 1.56E−04 1.04E−04 1.64E−04 1.68E−04 1.69E−04 8.30E−05 2.11E−04 2.44E−04 15.40 3.02E−04 1.11E−04 1.75E−04 1.01E−04 1.64E−04 1.70E−04 1.62E−04 5.60E−05 2.17E−04 2.39E−04 15.42 2.84E−04 1.13E−04 1.75E−04 1.07E−04 1.49E−04 1.52E−04 1.74E−04 7.10E−05 1.93E−04 2.42E−04 15.43 2.92E−04 1.15E−04 1.61E−04 1.09E−04 1.74E−04 1.53E−04 1.68E−04 6.00E−05 2.03E−04 2.28E−04 15.45 2.86E−04 9.50E−05 1.65E−04 1.10E−04 1.70E−04 1.65E−04 1.70E−04 6.40E−05 2.29E−04 2.33E−04 15.47 2.77E−04 1.13E−04 1.69E−04 1.11E−04 1.81E−04 1.63E−04 1.66E−04 6.20E−05 1.95E−04 2.23E−04 15.48 2.88E−04 1.01E−04 1.55E−04 1.11E−04 1.68E−04 1.60E−04 1.65E−04 7.30E−05 2.19E−04 2.38E−04 15.50 2.88E−04 9.40E−05 1.68E−04 1.25E−04 1.78E−04 1.63E−04 1.79E−04 7.60E−05 2.07E−04 2.24E−04 15.52 2.82E−04 1.11E−04 1.51E−04 1.01E−04 1.69E−04 1.53E−04 1.61E−04 7.10E−05 1.91E−04 2.34E−04 15.53 2.87E−04 9.60E−05 1.71E−04 1.26E−04 1.73E−04 1.50E−04 1.68E−04 5.70E−05 2.23E−04 2.17E−04 15.55 2.77E−04 1.09E−04 1.61E−04 9.70E−05 1.59E−04 1.72E−04 1.64E−04 6.70E−05 2.01E−04 2.20E−04 15.57 2.74E−04 1.05E−04 1.65E−04 1.06E−04 1.56E−04 1.45E−04 1.63E−04 7.00E−05 1.97E−04 2.33E−04 15.58 2.81E−04 1.02E−04 1.70E−04 1.06E−04 1.59E−04 1.61E−04 1.52E−04 6.40E−05 1.98E−04 2.29E−04 15.60 2.70E−04 1.01E−04 1.58E−04 1.06E−04 1.63E−04 1.56E−04 1.52E−04 6.30E−05 2.01E−04 2.34E−04 15.62 2.81E−04 9.40E−05 1.61E−04 1.05E−04 1.52E−04 1.56E−04 1.75E−04 5.80E−05 2.15E−04 2.14E−04 15.63 2.68E−04 9.20E−05 1.65E−04 1.02E−04 1.56E−04 1.60E−04 1.76E−04 6.90E−05 2.02E−04 2.23E−04 15.65 2.64E−04 9.10E−05 1.51E−04 9.60E−05 1.69E−04 1.42E−04 1.64E−04 5.40E−05 1.98E−04 2.33E−04 15.67 2.64E−04 1.03E−04 1.74E−04 1.07E−04 1.58E−04 1.52E−04 1.69E−04 7.00E−05 1.84E−04 2.31E−04 15.68 2.63E−04 8.40E−05 1.51E−04 9.30E−05 1.53E−04 1.52E−04 1.64E−04 5.20E−05 1.98E−04 2.29E−04 15.70 2.70E−04 1.06E−04 1.54E−04 1.02E−04 1.49E−04 1.41E−04 1.65E−04 5.00E−05 1.91E−04 2.11E−04 15.72 2.52E−04 8.10E−05 1.60E−04 9.30E−05 1.66E−04 1.48E−04 1.65E−04 6.20E−05 1.84E−04 2.33E−04 15.73 2.58E−04 9.30E−05 1.55E−04 1.04E−04 1.86E−04 1.51E−04 1.64E−04 5.80E−05 1.92E−04 2.20E−04 15.75 2.66E−04 8.90E−05 1.66E−04 9.90E−05 1.63E−04 1.45E−04 1.62E−04 6.00E−05 2.02E−04 2.28E−04 15.77 2.62E−04 7.50E−05 1.55E−04 9.20E−05 1.74E−04 1.39E−04 1.66E−04 5.50E−05 1.94E−04 2.33E−04 15.78 2.58E−04 9.60E−05 1.44E−04 9.90E−05 1.64E−04 1.50E−04 1.61E−04 6.00E−05 1.92E−04 2.13E−04 15.80 2.42E−04 7.70E−05 1.50E−04 9.40E−05 1.94E−04 1.39E−04 1.65E−04 3.50E−05 1.88E−04 2.32E−04 15.82 2.57E−04 9.50E−05 1.60E−04 9.20E−05 1.78E−04 1.45E−04 1.63E−04 5.00E−05 1.85E−04 2.21E−04 15.83 2.52E−04 8.30E−05 1.61E−04 1.03E−04 1.79E−04 1.53E−04 1.61E−04 4.40E−05 1.89E−04 2.24E−04 15.85 2.54E−04 9.40E−05 1.50E−04 1.02E−04 1.68E−04 1.42E−04 1.66E−04 6.70E−05 1.84E−04 2.44E−04 15.87 2.57E−04 8.50E−05 1.53E−04 1.09E−04 1.72E−04 1.50E−04 1.78E−04 5.10E−05 1.94E−04 2.34E−04 15.88 2.44E−04 8.90E−05 1.50E−04 1.05E−04 1.83E−04 1.58E−04 1.56E−04 5.40E−05 2.09E−04 2.33E−04 15.90 2.54E−04 8.20E−05 1.68E−04 1.26E−04 1.78E−04 1.50E−04 1.58E−04 4.30E−05 1.98E−04 2.20E−04 15.92 2.42E−04 7.90E−05 1.68E−04 1.13E−04 1.65E−04 1.42E−04 1.53E−04 4.80E−05 1.82E−04 2.29E−04 15.93 2.15E−04 7.90E−05 1.54E−04 1.24E−04 1.66E−04 1.44E−04 1.64E−04 6.50E−05 1.79E−04 2.25E−04 15.95 2.42E−04 8.70E−05 1.54E−04 1.17E−04 1.64E−04 1.49E−04 1.45E−04 4.60E−05 1.92E−04 2.12E−04 15.97 2.37E−04 7.40E−05 1.69E−04 1.06E−04 1.65E−04 1.34E−04 1.43E−04 5.00E−05 1.88E−04 2.19E−04 15.98 2.35E−04 7.00E−05 1.52E−04 1.10E−04 1.62E−04 1.45E−04 1.44E−04 4.50E−05 1.80E−04 2.34E−04 16.00 2.40E−04 8.90E−05 1.56E−04 1.04E−04 1.58E−04 1.33E−04 1.60E−04 5.30E−05 1.64E−04 2.31E−04 16.02 2.43E−04 7.90E−05 1.39E−04 1.12E−04 1.54E−04 1.30E−04 1.43E−04 6.20E−05 1.74E−04 2.15E−04 16.03 2.33E−04 6.70E−05 1.51E−04 8.00E−05 1.58E−04 1.44E−04 1.53E−04 4.70E−05 1.68E−04 2.21E−04 16.05 2.31E−04 8.30E−05 1.32E−04 8.60E−05 1.49E−04 1.25E−04 1.49E−04 3.10E−05 1.80E−04 2.14E−04 16.07 2.31E−04 7.10E−05 1.50E−04 1.06E−04 1.40E−04 1.44E−04 1.55E−04 4.40E−05 1.72E−04 2.18E−04 16.08 2.30E−04 7.50E−05 1.49E−04 7.20E−05 1.39E−04 1.33E−04 1.48E−04 3.80E−05 1.71E−04 2.07E−04 16.10 2.33E−04 6.40E−05 1.44E−04 9.60E−05 1.44E−04 1.17E−04 1.33E−04 4.10E−05 1.81E−04 2.04E−04 16.12 2.19E−04 7.50E−05 1.48E−04 8.50E−05 1.26E−04 1.25E−04 1.45E−04 2.10E−05 1.69E−04 2.07E−04 16.13 2.45E−04 6.40E−05 1.45E−04 7.90E−05 1.42E−04 1.23E−04 1.34E−04 3.70E−05 1.71E−04 2.13E−04 16.15 2.21E−04 6.70E−05 1.51E−04 6.10E−05 1.50E−04 1.22E−04 1.39E−04 2.60E−05 1.81E−04 2.17E−04 16.17 2.28E−04 6.90E−05 1.28E−04 8.70E−05 1.39E−04 1.07E−04 1.25E−04 3.30E−05 1.66E−04 2.08E−04 16.18 2.17E−04 9.50E−05 1.44E−04 8.40E−05 1.41E−04 1.20E−04 1.42E−04 3.60E−05 1.69E−04 2.03E−04 16.20 2.28E−04 7.30E−05 1.48E−04 9.20E−05 1.33E−04 1.22E−04 1.49E−04 2.80E−05 1.86E−04 2.12E−04 16.22 2.19E−04 6.50E−05 1.30E−04 7.40E−05 1.21E−04 1.11E−04 1.40E−04 3.30E−05 1.70E−04 2.33E−04 16.23 2.09E−04 6.60E−05 1.41E−04 7.60E−05 1.50E−04 1.23E−04 1.30E−04 2.30E−05 1.64E−04 2.14E−04 16.25 1.99E−04 7.90E−05 1.42E−04 7.70E−05 1.16E−04 1.17E−04 1.48E−04 3.50E−05 1.66E−04 2.07E−04 16.27 2.18E−04 5.80E−05 1.43E−04 7.60E−05 1.29E−04 1.25E−04 1.34E−04 3.30E−05 1.64E−04 2.12E−04 16.28 2.07E−04 6.40E−05 1.28E−04 7.40E−05 1.33E−04 1.13E−04 1.46E−04 3.10E−05 1.65E−04 2.13E−04 16.30 2.14E−04 6.60E−05 1.43E−04 8.60E−05 1.54E−04 1.09E−04 1.30E−04 3.00E−05 1.78E−04 2.04E−04 16.32 2.03E−04 5.60E−05 1.39E−04 7.30E−05 1.41E−04 1.17E−04 1.43E−04 2.30E−05 1.55E−04 2.10E−04 16.33 2.14E−04 6.60E−05 1.41E−04 8.00E−05 1.13E−04 1.03E−04 1.35E−04 2.40E−05 1.53E−04 2.08E−04 16.35 2.22E−04 6.50E−05 1.38E−04 7.90E−05 1.17E−04 1.20E−04 1.24E−04 3.10E−05 1.54E−04 2.02E−04 16.37 2.05E−04 5.00E−05 1.50E−04 7.20E−05 1.24E−04 1.17E−04 1.22E−04 4.10E−05 1.54E−04 2.09E−04 16.38 1.92E−04 6.40E−05 1.55E−04 8.20E−05 1.24E−04 1.03E−04 1.33E−04 2.70E−05 1.58E−04 2.09E−04 16.40 1.99E−04 6.00E−05 1.45E−04 6.60E−05 1.24E−04 1.28E−04 1.25E−04 1.50E−05 1.46E−04 2.13E−04 16.42 2.00E−04 6.00E−05 1.53E−04 5.30E−05 1.14E−04 1.06E−04 1.22E−04 1.50E−05 1.53E−04 2.11E−04 16.43 2.05E−04 5.50E−05 1.40E−04 5.50E−05 1.29E−04 9.10E−05 1.33E−04 2.00E−05 1.43E−04 2.10E−04 16.45 2.03E−04 4.40E−05 1.31E−04 6.50E−05 1.31E−04 1.04E−04 1.20E−04 2.50E−05 1.50E−04 2.15E−04 16.47 1.99E−04 5.00E−05 1.39E−04 6.10E−05 1.20E−04 1.00E−04 1.25E−04 3.00E−05 1.48E−04 2.27E−04 16.48 2.05E−04 4.80E−05 1.50E−04 7.10E−05 1.46E−04 9.90E−05 1.28E−04 3.00E−05 1.41E−04 2.08E−04 16.50 2.12E−04 5.10E−05 1.40E−04 5.50E−05 1.21E−04 1.07E−04 1.20E−04 1.80E−05 1.49E−04 2.15E−04 16.52 1.88E−04 5.00E−05 1.38E−04 5.50E−05 1.15E−04 1.13E−04 1.36E−04 1.60E−05 1.41E−04 2.12E−04 16.53 1.92E−04 5.40E−05 1.49E−04 5.10E−05 1.15E−04 1.11E−04 1.31E−04 2.60E−05 1.50E−04 2.01E−04 16.55 1.99E−04 5.60E−05 1.35E−04 5.60E−05 1.31E−04 1.13E−04 1.23E−04 2.40E−05 1.44E−04 2.20E−04 16.57 1.88E−04 6.90E−05 1.24E−04 5.40E−05 1.19E−04 1.22E−04 1.15E−04 3.00E−05 2.57E−04 2.17E−04 16.58 1.95E−04 5.30E−05 1.48E−04 4.80E−05 1.25E−04 1.17E−04 1.38E−04 2.00E−05 1.54E−04 2.04E−04 16.60 2.19E−04 5.80E−05 1.38E−04 5.00E−05 1.11E−04 1.04E−04 1.17E−04 1.10E−05 1.52E−04 2.05E−04 16.62 1.93E−04 6.50E−05 1.44E−04 5.50E−05 1.22E−04 1.05E−04 1.29E−04 1.60E−05 1.42E−04 2.15E−04 16.63 1.81E−04 3.60E−05 1.38E−04 5.40E−05 1.34E−04 1.10E−04 1.60E−04 1.20E−05 1.48E−04 2.04E−04 16.65 1.78E−04 4.60E−05 1.42E−04 5.80E−05 1.31E−04 1.12E−04 1.14E−04 3.40E−05 1.43E−04 2.07E−04 16.67 1.99E−04 4.40E−05 1.51E−04 7.30E−05 1.14E−04 1.06E−04 1.24E−04 2.10E−05 1.53E−04 2.00E−04 16.68 1.92E−04 5.00E−05 1.32E−04 6.50E−05 1.10E−04 8.40E−05 1.31E−04 1.30E−05 1.41E−04 2.03E−04 16.70 1.93E−04 4.10E−05 1.38E−04 6.10E−05 1.12E−04 9.60E−05 1.10E−04 2.00E−06 1.42E−04 1.99E−04 16.72 2.02E−04 5.80E−05 1.44E−04 6.40E−05 1.16E−04 1.07E−04 1.21E−04 1.20E−05 1.55E−04 1.97E−04 16.73 1.85E−04 5.50E−05 1.44E−04 5.50E−05 1.11E−04 1.01E−04 1.03E−04 1.70E−05 1.46E−04 2.13E−04 16.75 2.01E−04 2.50E−05 1.38E−04 5.60E−05 1.24E−04 1.11E−04 1.20E−04 1.40E−05 1.32E−04 2.08E−04 16.77 1.81E−04 5.20E−05 1.50E−04 4.40E−05 1.07E−04 1.16E−04 1.06E−04 0.00E+00 1.28E−04 1.99E−04 16.78 1.95E−04 6.40E−05 1.46E−04 5.30E−05 1.13E−04 1.07E−04 1.10E−04 1.70E−05 1.36E−04 2.03E−04 16.80 1.94E−04 4.30E−05 1.41E−04 4.40E−05 1.14E−04 9.40E−05 1.11E−04 1.00E−05 1.51E−04 2.04E−04 16.82 1.94E−04 5.50E−05 1.38E−04 4.30E−05 1.17E−04 1.00E−04 1.05E−04 1.30E−05 1.35E−04 2.04E−04 16.83 1.75E−04 6.00E−05 1.28E−04 4.00E−05 1.09E−04 9.90E−05 1.06E−04 1.30E−05 1.56E−04 2.12E−04 16.85 2.01E−04 4.60E−05 1.55E−04 3.10E−05 1.09E−04 9.70E−05 1.09E−04 3.00E−06 1.41E−04 1.94E−04 16.87 1.82E−04 6.30E−05 1.43E−04 5.10E−05 1.26E−04 9.10E−05 1.14E−04 1.60E−05 1.30E−04 1.94E−04 16.88 2.04E−04 4.60E−05 1.33E−04 3.70E−05 1.06E−04 1.00E−04 1.11E−04 1.10E−05 1.44E−04 1.83E−04 16.90 1.85E−04 4.10E−05 1.46E−04 3.70E−05 1.12E−04 9.20E−05 9.90E−05 6.00E−06 1.42E−04 1.98E−04 16.92 1.78E−04 3.80E−05 1.44E−04 4.20E−05 1.11E−04 1.09E−04 9.10E−05 3.00E−05 1.31E−04 1.90E−04 16.93 1.93E−04 4.60E−05 1.39E−04 4.00E−05 8.90E−05 1.03E−04 1.20E−04 2.80E−05 1.36E−04 1.91E−04 16.95 1.94E−04 3.80E−05 1.50E−04 4.60E−05 1.02E−04 8.70E−05 1.07E−04 1.50E−05 1.30E−04 1.98E−04 16.97 1.85E−04 4.60E−05 1.38E−04 4.10E−05 1.06E−04 9.70E−05 1.03E−04 2.50E−05 1.32E−04 1.84E−04 16.98 1.85E−04 3.10E−05 1.29E−04 2.80E−05 1.23E−04 9.10E−05 1.06E−04 8.00E−06 1.25E−04 1.99E−04 17.00 2.01E−04 3.40E−05 1.46E−04 3.50E−05 1.00E−04 7.70E−05 9.70E−05 2.00E−05 1.29E−04 1.88E−04 17.02 1.90E−04 4.80E−05 1.40E−04 3.60E−05 1.12E−04 9.60E−05 1.07E−04 1.70E−05 1.30E−04 1.91E−04 17.03 2.08E−04 4.70E−05 1.55E−04 3.80E−05 1.10E−04 9.40E−05 1.11E−04 2.00E−06 1.35E−04 1.83E−04 17.05 1.83E−04 3.50E−05 1.33E−04 6.10E−05 8.50E−05 8.70E−05 1.10E−04 5.00E−06 1.29E−04 1.85E−04 17.07 1.76E−04 3.60E−05 1.33E−04 3.20E−05 1.03E−04 9.20E−05 9.70E−05 2.10E−05 1.25E−04 1.93E−04 17.08 1.86E−04 5.30E−05 1.39E−04 3.30E−05 1.20E−04 9.40E−05 1.04E−04 2.20E−05 1.25E−04 1.92E−04 17.10 1.85E−04 4.80E−05 1.48E−04 4.30E−05 1.02E−04 8.50E−05 8.30E−05 1.50E−05 1.49E−04 1.91E−04 17.12 1.84E−04 4.80E−05 1.43E−04 5.30E−05 1.15E−04 8.00E−05 8.90E−05 7.00E−06 1.32E−04 1.85E−04 17.13 1.80E−04 4.40E−05 1.44E−04 3.60E−05 1.01E−04 8.30E−05 7.40E−05 7.00E−06 1.21E−04 1.90E−04 17.15 1.95E−04 4.80E−05 1.38E−04 4.30E−05 1.02E−04 9.30E−05 9.90E−05 −7.00E−06 1.45E−04 1.89E−04 17.17 1.83E−04 4.40E−05 1.40E−04 3.60E−05 1.03E−04 9.20E−05 9.00E−05 1.00E−06 1.20E−04 1.93E−04 17.18 1.95E−04 4.20E−05 1.23E−04 3.30E−05 9.20E−05 9.70E−05 1.19E−04 −2.00E−06 1.26E−04 1.82E−04 17.20 1.91E−04 4.20E−05 1.40E−04 3.80E−05 1.19E−04 8.10E−05 1.01E−04 −1.00E−05 1.25E−04 1.92E−04 17.22 1.91E−04 5.60E−05 1.44E−04 4.40E−05 1.10E−04 1.01E−04 1.05E−04 5.00E−06 1.25E−04 1.76E−04 17.23 1.93E−04 3.20E−05 1.26E−04 4.00E−05 1.16E−04 8.90E−05 9.60E−05 4.00E−06 1.13E−04 1.97E−04 17.25 1.91E−04 4.50E−05 1.44E−04 5.50E−05 1.12E−04 9.40E−05 1.07E−04 5.00E−06 1.28E−04 1.83E−04 17.27 1.80E−04 4.80E−05 1.40E−04 4.20E−05 1.09E−04 8.90E−05 9.00E−05 −7.00E−06 1.45E−04 1.75E−04 17.28 1.84E−04 3.50E−05 1.41E−04 6.60E−05 1.23E−04 9.10E−05 1.05E−04 3.00E−06 1.28E−04 1.88E−04 17.30 2.03E−04 3.20E−05 1.48E−04 7.00E−05 1.12E−04 9.70E−05 7.70E−05 8.00E−06 1.24E−04 1.89E−04 17.32 2.00E−04 3.20E−05 1.44E−04 6.40E−05 1.32E−04 9.50E−05 1.00E−04 1.00E−06 1.42E−04 1.80E−04 17.33 2.00E−04 4.30E−05 1.43E−04 9.00E−05 1.36E−04 9.20E−05 9.30E−05 2.00E−06 1.21E−04 1.98E−04 17.35 1.94E−04 3.30E−05 1.52E−04 1.04E−04 1.22E−04 1.07E−04 9.60E−05 6.00E−06 1.25E−04 1.80E−04 17.37 1.91E−04 3.10E−05 1.42E−04 9.10E−05 1.46E−04 1.17E−04 9.30E−05 8.00E−06 1.24E−04 1.90E−04 17.38 1.86E−04 3.00E−05 1.33E−04 1.36E−04 1.46E−04 1.11E−04 1.07E−04 1.30E−05 1.23E−04 1.82E−04 17.40 1.88E−04 3.70E−05 1.38E−04 1.30E−04 1.66E−04 1.11E−04 9.10E−05 3.00E−06 1.31E−04 1.86E−04 17.42 2.01E−04 3.30E−05 1.39E−04 1.33E−04 1.60E−04 1.05E−04 9.40E−05 1.50E−05 1.28E−04 1.93E−04 17.43 1.73E−04 4.10E−05 1.36E−04 1.41E−04 1.43E−04 1.16E−04 9.70E−05 1.00E−05 1.23E−04 1.93E−04 17.45 1.88E−04 4.00E−05 1.26E−04 1.49E−04 1.45E−04 1.17E−04 9.90E−05 6.00E−06 1.15E−04 1.92E−04 17.47 1.81E−04 4.00E−05 1.35E−04 1.28E−04 1.19E−04 1.17E−04 9.10E−05 5.00E−06 1.03E−04 1.81E−04 17.48 1.74E−04 3.40E−05 1.39E−04 1.20E−04 1.28E−04 1.10E−04 9.30E−05 5.00E−06 1.15E−04 1.74E−04 17.50 1.78E−04 4.10E−05 1.32E−04 1.14E−04 1.14E−04 1.07E−04 1.00E−04 1.00E−06 1.14E−04 1.88E−04 17.52 1.85E−04 3.70E−05 1.36E−04 1.11E−04 1.19E−04 1.15E−04 9.70E−05 2.00E−06 1.23E−04 1.83E−04 17.53 1.83E−04 3.30E−05 1.40E−04 9.10E−05 1.15E−04 1.07E−04 9.60E−05 0.00E+00 1.13E−04 1.83E−04 17.55 1.88E−04 3.80E−05 1.29E−04 8.40E−05 1.17E−04 9.60E−05 8.40E−05 3.00E−06 1.17E−04 1.84E−04 17.57 1.80E−04 4.40E−05 1.39E−04 8.90E−05 9.20E−05 8.10E−05 8.20E−05 5.00E−06 1.19E−04 1.79E−04 17.58 1.82E−04 3.50E−05 1.33E−04 7.40E−05 1.02E−04 9.20E−05 9.20E−05 −7.00E−06 1.24E−04 1.90E−04 17.60 1.78E−04 3.50E−05 1.17E−04 7.20E−05 9.50E−05 7.90E−05 9.70E−05 1.00E−05 1.22E−04 1.76E−04 17.62 1.79E−04 3.50E−05 1.21E−04 5.40E−05 8.60E−05 8.10E−05 9.00E−05 1.40E−05 1.09E−04 1.84E−04 17.63 1.72E−04 4.70E−05 1.11E−04 5.60E−05 9.40E−05 7.60E−05 6.20E−05 1.00E−05 1.04E−04 1.92E−04 17.65 1.80E−04 3.50E−05 1.23E−04 7.40E−05 9.00E−05 8.50E−05 9.00E−05 −7.00E−06 1.29E−04 1.65E−04 17.67 1.88E−04 3.40E−05 1.21E−04 5.20E−05 9.90E−05 9.00E−05 8.90E−05 8.00E−06 1.16E−04 1.61E−04 17.68 1.75E−04 2.60E−05 1.32E−04 4.60E−05 9.10E−05 8.40E−05 7.50E−05 3.00E−06 1.01E−04 1.72E−04 17.70 1.76E−04 4.20E−05 1.19E−04 5.70E−05 8.10E−05 8.90E−05 8.60E−05 1.00E−06 1.04E−04 1.69E−04 17.72 1.55E−04 3.30E−05 1.31E−04 4.30E−05 8.30E−05 8.20E−05 8.70E−05 6.00E−06 1.15E−04 1.71E−04 17.73 1.76E−04 4.00E−05 1.24E−04 4.00E−05 9.20E−05 8.50E−05 8.60E−05 1.20E−05 1.11E−04 1.75E−04 17.75 1.59E−04 4.60E−05 1.20E−04 4.00E−05 9.90E−05 8.00E−05 7.90E−05 −8.00E−06 1.05E−04 1.64E−04 17.77 1.69E−04 3.80E−05 1.17E−04 3.00E−05 9.20E−05 7.70E−05 9.10E−05 3.00E−06 1.09E−04 1.73E−04 17.78 1.71E−04 4.00E−05 1.15E−04 4.20E−05 8.50E−05 7.90E−05 7.40E−05 −4.00E−06 1.14E−04 1.71E−04 17.80 1.82E−04 4.70E−05 1.24E−04 4.20E−05 1.04E−04 9.20E−05 6.70E−05 1.50E−05 1.05E−04 1.73E−04 17.82 1.75E−04 3.30E−05 1.09E−04 4.20E−05 9.70E−05 8.10E−05 6.90E−05 −3.00E−06 1.16E−04 1.73E−04 17.83 1.53E−04 3.60E−05 1.21E−04 4.40E−05 7.20E−05 8.50E−05 7.70E−05 −1.00E−06 1.07E−04 1.78E−04 17.85 1.66E−04 3.80E−05 1.22E−04 3.60E−05 7.90E−05 9.20E−05 8.90E−05 5.00E−06 1.06E−04 1.73E−04 17.87 1.63E−04 3.40E−05 1.12E−04 5.30E−05 7.50E−05 7.90E−05 9.30E−05 −1.70E−05 1.16E−04 1.80E−04 17.88 1.56E−04 4.00E−05 1.11E−04 8.00E−05 7.30E−05 8.30E−05 7.20E−05 0.00E+00 1.16E−04 1.73E−04 17.90 1.60E−04 3.80E−05 1.10E−04 4.20E−05 7.10E−05 8.10E−05 7.40E−05 −6.00E−06 1.13E−04 1.86E−04 17.92 1.53E−04 2.30E−05 9.30E−05 4.30E−05 8.00E−05 9.10E−05 7.40E−05 0.00E+00 1.10E−04 1.66E−04 17.93 1.50E−04 3.40E−05 1.03E−04 3.60E−05 8.10E−05 8.00E−05 7.30E−05 −5.00E−06 1.07E−04 1.84E−04 17.95 1.51E−04 3.50E−05 1.07E−04 3.20E−05 6.60E−05 7.40E−05 7.40E−05 −4.00E−06 1.02E−04 1.71E−04 17.97 1.68E−04 5.50E−05 9.30E−05 5.30E−05 8.70E−05 9.90E−05 8.50E−05 −1.60E−05 1.17E−04 1.83E−04 17.98 1.60E−04 2.50E−05 1.24E−04 3.80E−05 7.40E−05 7.70E−05 7.00E−05 −7.00E−06 1.23E−04 1.59E−04 18.00 1.55E−04 3.60E−05 1.14E−04 4.30E−05 9.30E−05 8.20E−05 8.20E−05 −8.00E−06 1.05E−04 1.66E−04 18.02 1.51E−04 4.20E−05 1.09E−04 2.60E−05 7.20E−05 8.00E−05 6.60E−05 −3.00E−06 1.10E−04 1.76E−04 18.03 1.60E−04 1.80E−05 1.07E−04 2.70E−05 8.30E−05 7.90E−05 6.70E−05 1.10E−05 1.10E−04 1.74E−04 18.05 1.48E−04 4.40E−05 1.14E−04 3.60E−05 6.20E−05 6.90E−05 8.00E−05 −1.60E−05 9.70E−05 1.69E−04 18.07 1.55E−04 4.20E−05 1.09E−04 3.20E−05 7.20E−05 8.20E−05 6.70E−05 −4.00E−06 1.06E−04 1.66E−04 18.08 1.36E−04 3.80E−05 1.10E−04 3.20E−05 7.50E−05 8.10E−05 7.10E−05 1.10E−05 1.15E−04 1.81E−04 18.10 1.42E−04 1.70E−05 1.06E−04 4.00E−05 7.20E−05 6.30E−05 7.40E−05 −8.00E−06 1.17E−04 1.78E−04 18.12 1.54E−04 3.60E−05 8.20E−05 4.30E−05 8.10E−05 9.50E−05 7.70E−05 −1.30E−05 8.50E−05 1.85E−04 18.13 1.43E−04 3.40E−05 1.09E−04 4.80E−05 6.90E−05 6.70E−05 7.40E−05 −1.30E−05 1.03E−04 1.73E−04 18.15 1.55E−04 2.60E−05 1.03E−04 3.80E−05 6.40E−05 6.70E−05 7.40E−05 −5.00E−06 1.16E−04 1.56E−04 18.17 1.45E−04 4.00E−05 1.07E−04 4.20E−05 6.40E−05 7.40E−05 7.20E−05 −3.00E−06 1.11E−04 1.78E−04 18.18 1.53E−04 3.80E−05 1.00E−04 4.80E−05 7.60E−05 6.90E−05 6.40E−05 8.00E−06 8.50E−05 1.65E−04 18.20 1.62E−04 3.10E−05 1.10E−04 3.70E−05 7.00E−05 9.00E−05 6.30E−05 6.00E−06 1.07E−04 1.71E−04 18.22 1.54E−04 2.80E−05 9.40E−05 4.80E−05 8.20E−05 7.00E−05 6.90E−05 −3.00E−06 1.06E−04 1.71E−04 18.23 1.36E−04 2.50E−05 9.70E−05 4.00E−05 7.90E−05 6.60E−05 5.80E−05 −4.00E−06 1.00E−04 1.70E−04 18.25 1.42E−04 3.00E−05 1.09E−04 3.30E−05 7.90E−05 9.40E−05 7.40E−05 −5.00E−06 1.07E−04 1.83E−04 18.27 1.53E−04 3.30E−05 1.03E−04 2.30E−05 6.60E−05 7.00E−05 5.30E−05 1.50E−05 1.09E−04 1.82E−04 18.28 1.45E−04 1.60E−05 9.40E−05 4.40E−05 7.50E−05 6.40E−05 5.70E−05 −2.00E−06 1.13E−04 1.89E−04 18.30 1.52E−04 4.10E−05 8.30E−05 5.50E−05 7.40E−05 7.50E−05 7.20E−05 −1.00E−06 1.12E−04 1.73E−04 18.32 1.51E−04 2.50E−05 9.00E−05 4.60E−05 6.90E−05 9.00E−05 5.00E−05 −6.00E−06 1.23E−04 1.69E−04 18.33 1.44E−04 4.80E−05 9.90E−05 2.80E−05 6.10E−05 7.10E−05 5.60E−05 1.00E−05 1.02E−04 1.78E−04 18.35 1.42E−04 2.80E−05 1.03E−04 3.70E−05 7.20E−05 7.20E−05 6.60E−05 −6.00E−06 1.11E−04 1.64E−04 18.37 1.40E−04 2.60E−05 9.60E−05 3.20E−05 7.20E−05 7.00E−05 6.20E−05 −1.00E−05 1.02E−04 1.97E−04 18.38 1.51E−04 3.40E−05 9.40E−05 3.50E−05 8.10E−05 7.20E−05 7.70E−05 −1.60E−05 1.03E−04 1.80E−04 18.40 1.43E−04 3.20E−05 9.50E−05 3.50E−05 6.50E−05 7.00E−05 5.70E−05 −1.60E−05 1.16E−04 1.84E−04 18.42 1.48E−04 2.80E−05 9.10E−05 3.50E−05 6.40E−05 8.40E−05 6.30E−05 −3.00E−06 9.70E−05 1.74E−04 18.43 1.32E−04 3.40E−05 1.00E−04 3.40E−05 6.50E−05 8.40E−05 5.00E−05 −5.00E−06 1.10E−04 1.91E−04 18.45 1.53E−04 3.50E−05 1.03E−04 4.30E−05 6.40E−05 7.20E−05 6.20E−05 −1.50E−05 1.07E−04 1.76E−04 18.47 1.41E−04 3.10E−05 9.50E−05 4.20E−05 7.00E−05 8.10E−05 7.90E−05 −2.20E−05 1.04E−04 1.76E−04 18.48 1.26E−04 4.70E−05 9.00E−05 4.40E−05 6.10E−05 8.40E−05 7.70E−05 −7.00E−06 1.03E−04 1.79E−04 18.50 1.36E−04 4.20E−05 9.90E−05 1.01E−04 7.60E−05 7.50E−05 5.30E−05 7.00E−06 1.05E−04 1.83E−04 18.52 1.38E−04 3.40E−05 1.01E−04 4.50E−05 6.90E−05 7.00E−05 5.70E−05 −5.00E−06 9.90E−05 1.89E−04 18.53 1.26E−04 3.30E−05 9.90E−05 6.20E−05 7.00E−05 7.40E−05 6.40E−05 −2.10E−05 1.09E−04 1.61E−04 18.55 1.24E−04 4.60E−05 8.90E−05 4.10E−05 7.10E−05 5.60E−05 5.10E−05 −1.40E−05 1.04E−04 1.80E−04 18.57 1.36E−04 4.50E−05 9.40E−05 4.70E−05 6.50E−05 7.10E−05 5.80E−05 −3.00E−06 1.28E−04 1.84E−04 18.58 1.33E−04 4.00E−05 1.06E−04 5.40E−05 6.10E−05 7.50E−05 4.70E−05 −6.00E−06 1.11E−04 1.90E−04 18.60 1.29E−04 4.10E−05 1.01E−04 4.60E−05 7.50E−05 9.00E−05 7.60E−05 −6.00E−06 1.02E−04 1.80E−04 18.62 1.40E−04 4.20E−05 1.06E−04 3.70E−05 6.30E−05 7.10E−05 5.60E−05 −4.00E−06 1.03E−04 1.74E−04 18.63 1.34E−04 3.10E−05 9.60E−05 3.70E−05 5.80E−05 7.60E−05 5.00E−05 −2.00E−06 1.11E−04 1.90E−04 18.65 1.26E−04 3.50E−05 8.90E−05 3.30E−05 5.70E−05 7.00E−05 4.30E−05 −1.00E−05 1.03E−04 1.90E−04 18.67 1.40E−04 3.80E−05 9.00E−05 3.40E−05 5.00E−05 6.20E−05 5.10E−05 −4.00E−06 1.05E−04 1.83E−04 18.68 2.31E−04 4.30E−05 8.40E−05 4.80E−05 7.00E−05 7.40E−05 4.70E−05 −5.00E−06 9.60E−05 1.75E−04 18.70 1.24E−04 4.00E−05 9.40E−05 3.50E−05 7.10E−05 7.60E−05 6.30E−05 −1.30E−05 9.90E−05 1.83E−04 18.72 1.41E−04 4.60E−05 1.12E−04 4.10E−05 7.40E−05 6.50E−05 5.80E−05 −5.00E−06 1.10E−04 2.03E−04 18.73 1.56E−04 3.70E−05 9.40E−05 5.20E−05 5.60E−05 7.60E−05 6.40E−05 −3.00E−06 1.07E−04 1.66E−04 18.75 1.21E−04 2.60E−05 1.32E−04 3.80E−05 5.70E−05 5.50E−05 6.40E−05 −8.00E−06 1.00E−04 1.80E−04 18.77 1.13E−04 4.40E−05 8.90E−05 5.50E−05 7.10E−05 8.70E−05 5.10E−05 −1.00E−05 1.12E−04 1.83E−04 18.78 1.33E−04 5.60E−05 1.03E−04 4.70E−05 6.00E−05 7.20E−05 4.30E−05 −1.50E−05 9.60E−05 1.66E−04 18.80 1.38E−04 6.30E−05 8.30E−05 4.60E−05 7.50E−05 6.90E−05 5.20E−05 −1.50E−05 1.01E−04 1.86E−04 18.82 1.19E−04 6.50E−05 7.40E−05 6.10E−05 7.20E−05 5.30E−05 5.20E−05 −1.00E−05 1.05E−04 1.84E−04 18.83 1.34E−04 5.40E−05 8.70E−05 3.50E−05 6.20E−05 6.50E−05 5.80E−05 −2.10E−05 9.90E−05 1.81E−04 18.85 1.24E−04 5.00E−05 1.14E−04 4.10E−05 5.40E−05 5.30E−05 6.40E−05 −3.00E−06 9.60E−05 1.83E−04 18.87 1.21E−04 5.50E−05 9.50E−05 2.80E−05 6.60E−05 5.30E−05 5.30E−05 −1.70E−05 9.70E−05 1.70E−04 18.88 1.38E−04 5.40E−05 9.60E−05 4.40E−05 5.00E−05 7.70E−05 6.00E−05 −6.00E−06 1.07E−04 1.76E−04 18.90 1.33E−04 4.50E−05 8.60E−05 4.50E−05 5.80E−05 8.00E−05 5.30E−05 7.00E−06 9.20E−05 1.85E−04 18.92 1.43E−04 6.10E−05 8.20E−05 3.10E−05 5.10E−05 6.90E−05 4.30E−05 −8.00E−06 1.07E−04 1.83E−04 18.93 1.22E−04 5.60E−05 8.50E−05 4.10E−05 5.10E−05 5.60E−05 5.40E−05 −1.10E−05 9.50E−05 1.78E−04 18.95 1.16E−04 7.40E−05 9.70E−05 5.80E−05 7.00E−05 7.30E−05 7.90E−05 −1.70E−05 9.70E−05 1.76E−04 18.97 1.35E−04 4.70E−05 7.70E−05 4.00E−05 5.80E−05 8.10E−05 5.80E−05 −1.30E−05 1.09E−04 1.92E−04 18.98 1.28E−04 6.30E−05 8.50E−05 5.10E−05 6.20E−05 7.90E−05 6.70E−05 −1.20E−05 1.07E−04 1.73E−04 19.00 1.14E−04 6.50E−05 8.60E−05 3.20E−05 8.00E−05 6.90E−05 4.80E−05 −2.10E−05 1.17E−04 1.72E−04 19.02 1.21E−04 6.60E−05 8.70E−05 4.50E−05 5.60E−05 6.40E−05 5.20E−05 −2.70E−05 1.05E−04 1.79E−04 19.03 1.05E−04 7.10E−05 7.90E−05 6.30E−05 4.70E−05 8.10E−05 6.10E−05 −1.00E−05 1.06E−04 1.85E−04 19.05 1.14E−04 5.80E−05 8.00E−05 6.10E−05 5.10E−05 7.00E−05 6.70E−05 −1.30E−05 1.14E−04 1.72E−04 19.07 1.33E−04 7.30E−05 7.40E−05 4.10E−05 6.10E−05 6.10E−05 5.80E−05 −1.60E−05 1.01E−04 1.86E−04 19.08 1.28E−04 5.80E−05 9.30E−05 3.80E−05 6.40E−05 7.00E−05 5.60E−05 −2.70E−05 1.02E−04 1.81E−04 19.10 1.26E−04 7.00E−05 8.10E−05 4.40E−05 6.00E−05 6.70E−05 5.40E−05 −7.00E−06 1.03E−04 1.70E−04 19.12 1.24E−04 5.70E−05 9.70E−05 3.10E−05 6.20E−05 7.70E−05 6.00E−05 −3.20E−05 1.03E−04 1.82E−04 19.13 1.15E−04 6.60E−05 7.00E−05 5.10E−05 8.40E−05 6.10E−05 5.70E−05 −2.20E−05 1.17E−04 1.98E−04 19.15 1.16E−04 6.60E−05 8.10E−05 4.10E−05 5.20E−05 5.70E−05 5.00E−05 −2.60E−05 1.15E−04 1.83E−04 19.17 1.21E−04 7.20E−05 8.30E−05 4.40E−05 5.80E−05 6.30E−05 4.30E−05 −1.50E−05 1.05E−04 1.79E−04 19.18 1.15E−04 6.70E−05 8.60E−05 5.10E−05 4.20E−05 7.90E−05 6.10E−05 −6.00E−06 1.07E−04 1.66E−04 19.20 1.34E−04 6.70E−05 8.60E−05 4.80E−05 6.40E−05 5.60E−05 5.70E−05 −1.50E−05 9.90E−05 1.73E−04 19.22 1.14E−04 6.70E−05 8.50E−05 4.20E−05 6.50E−05 6.30E−05 5.20E−05 −2.60E−05 1.05E−04 1.89E−04 19.23 1.29E−04 7.50E−05 8.50E−05 4.20E−05 5.20E−05 7.20E−05 5.30E−05 −1.60E−05 9.30E−05 1.78E−04 19.25 1.14E−04 7.90E−05 8.00E−05 3.80E−05 4.60E−05 7.50E−05 6.70E−05 −2.80E−05 9.30E−05 1.76E−04 19.27 1.15E−04 6.90E−05 8.20E−05 4.70E−05 5.60E−05 5.70E−05 5.70E−05 −2.10E−05 1.00E−04 1.89E−04 19.28 1.13E−04 7.60E−05 8.10E−05 4.60E−05 5.00E−05 8.10E−05 5.60E−05 −2.30E−05 1.02E−04 1.61E−04 19.30 1.26E−04 8.40E−05 8.30E−05 3.20E−05 5.50E−05 5.70E−05 5.40E−05 −1.70E−05 1.10E−04 1.74E−04 19.32 1.09E−04 5.50E−05 8.60E−05 3.00E−05 5.60E−05 7.20E−05 6.40E−05 −2.20E−05 1.10E−04 1.71E−04 19.33 1.15E−04 4.80E−05 7.60E−05 3.50E−05 6.10E−05 5.30E−05 4.80E−05 −3.20E−05 1.15E−04 1.58E−04 19.35 1.16E−04 6.60E−05 8.90E−05 4.10E−05 5.30E−05 6.50E−05 5.10E−05 −2.10E−05 1.03E−04 1.79E−04 19.37 1.23E−04 5.50E−05 8.70E−05 5.00E−05 4.40E−05 5.60E−05 6.40E−05 1.40E−05 9.90E−05 1.73E−04 19.38 1.44E−04 7.50E−05 8.00E−05 5.40E−05 4.80E−05 6.00E−05 4.30E−05 −1.20E−05 9.30E−05 1.71E−04 19.40 1.23E−04 5.60E−05 9.60E−05 3.10E−05 5.50E−05 6.30E−05 6.10E−05 −1.10E−05 1.05E−04 1.75E−04 19.42 1.24E−04 8.10E−05 6.50E−05 3.60E−05 4.00E−05 6.30E−05 4.50E−05 −1.50E−05 1.02E−04 1.71E−04 19.43 1.09E−04 6.30E−05 7.20E−05 4.30E−05 4.80E−05 7.20E−05 5.20E−05 −1.40E−05 9.30E−05 1.84E−04 19.45 1.19E−04 7.90E−05 8.00E−05 5.40E−05 5.80E−05 5.80E−05 5.50E−05 −1.30E−05 1.05E−04 1.79E−04 19.47 1.09E−04 7.10E−05 7.40E−05 3.80E−05 7.10E−05 7.20E−05 5.60E−05 −7.00E−06 1.01E−04 1.84E−04 19.48 1.23E−04 5.80E−05 7.00E−05 3.20E−05 5.10E−05 5.50E−05 5.70E−05 −1.50E−05 1.00E−04 1.60E−04 19.50 1.15E−04 7.30E−05 7.40E−05 5.70E−05 6.60E−05 6.30E−05 4.50E−05 −2.20E−05 1.01E−04 1.68E−04 19.52 1.21E−04 6.10E−05 8.00E−05 3.50E−05 6.30E−05 6.20E−05 6.50E−05 −2.70E−05 9.70E−05 1.84E−04 19.53 1.25E−04 7.30E−05 7.40E−05 4.50E−05 7.60E−05 7.60E−05 7.60E−05 −1.50E−05 9.90E−05 1.78E−04 19.55 1.07E−04 7.90E−05 9.10E−05 4.10E−05 7.10E−05 5.50E−05 4.40E−05 −1.40E−05 9.30E−05 1.71E−04 19.57 1.19E−04 7.50E−05 8.70E−05 5.50E−05 5.80E−05 7.00E−05 6.40E−05 −1.70E−05 9.50E−05 1.62E−04 19.58 1.06E−04 7.60E−05 7.50E−05 3.70E−05 6.40E−05 6.70E−05 6.70E−05 −1.70E−05 1.04E−04 1.71E−04 19.60 1.23E−04 6.90E−05 7.90E−05 4.30E−05 6.60E−05 7.70E−05 6.50E−05 −1.20E−05 9.50E−05 1.69E−04 19.62 1.12E−04 8.10E−05 8.30E−05 3.20E−05 6.60E−05 6.70E−05 6.40E−05 −1.70E−05 1.04E−04 1.82E−04 19.63 9.30E−05 9.70E−05 7.90E−05 3.80E−05 5.60E−05 8.30E−05 5.80E−05 −1.80E−05 1.00E−04 2.29E−04 19.65 1.09E−04 7.10E−05 6.10E−05 3.40E−05 5.40E−05 6.60E−05 6.30E−05 −1.30E−05 9.30E−05 1.73E−04 19.67 1.15E−04 7.30E−05 8.10E−05 4.50E−05 7.20E−05 7.90E−05 5.30E−05 −1.00E−05 1.01E−04 1.78E−04 19.68 1.14E−04 7.60E−05 7.20E−05 3.80E−05 5.40E−05 8.50E−05 6.20E−05 −1.80E−05 9.10E−05 1.78E−04 19.70 9.90E−05 7.90E−05 9.10E−05 4.40E−05 6.70E−05 6.30E−05 6.50E−05 −7.00E−06 9.20E−05 1.75E−04 19.72 1.14E−04 7.60E−05 6.40E−05 4.20E−05 6.50E−05 7.20E−05 5.70E−05 −1.80E−05 9.40E−05 1.65E−04 19.73 1.00E−04 7.20E−05 8.50E−05 5.20E−05 8.00E−05 6.70E−05 5.30E−05 −1.30E−05 9.00E−05 1.75E−04 19.75 1.02E−04 7.90E−05 7.70E−05 2.70E−05 7.00E−05 8.00E−05 6.20E−05 −2.50E−05 1.05E−04 1.76E−04 19.77 9.70E−05 8.60E−05 5.60E−05 4.10E−05 7.10E−05 9.10E−05 5.20E−05 −1.30E−05 1.06E−04 1.71E−04 19.78 1.15E−04 8.50E−05 7.50E−05 3.20E−05 8.10E−05 7.20E−05 5.30E−05 −8.00E−06 9.90E−05 1.63E−04 19.80 1.13E−04 7.60E−05 8.20E−05 3.20E−05 9.20E−05 9.00E−05 5.60E−05 −1.70E−05 8.70E−05 1.69E−04 19.82 1.16E−04 7.20E−05 7.00E−05 3.50E−05 7.70E−05 8.20E−05 5.00E−05 −2.30E−05 1.01E−04 1.73E−04 19.83 1.23E−04 6.50E−05 6.10E−05 3.00E−05 7.60E−05 8.30E−05 4.70E−05 −1.60E−05 9.30E−05 1.59E−04 19.85 1.26E−04 7.70E−05 7.10E−05 3.00E−05 8.00E−05 7.70E−05 4.70E−05 −1.80E−05 1.02E−04 1.73E−04 19.87 1.23E−04 6.60E−05 8.00E−05 4.60E−05 7.50E−05 7.70E−05 5.80E−05 −8.00E−06 1.00E−04 1.74E−04 19.88 1.24E−04 7.40E−05 7.10E−05 1.20E−05 7.60E−05 7.60E−05 6.00E−05 −1.30E−05 9.10E−05 1.74E−04 19.90 1.15E−04 6.10E−05 6.60E−05 3.20E−05 7.70E−05 7.40E−05 5.40E−05 −2.30E−05 8.70E−05 2.01E−04 19.92 1.34E−04 6.30E−05 6.90E−05 3.80E−05 8.40E−05 7.40E−05 5.80E−05 −2.30E−05 1.03E−04 1.62E−04 19.93 1.26E−04 8.70E−05 8.30E−05 3.00E−05 8.20E−05 8.60E−05 5.10E−05 −1.50E−05 1.04E−04 1.55E−04 19.95 1.01E−04 6.50E−05 6.20E−05 2.10E−05 7.30E−05 9.20E−05 5.70E−05 −1.10E−05 1.04E−04 1.78E−04 19.97 1.10E−04 8.10E−05 8.10E−05 3.20E−05 7.10E−05 8.50E−05 5.00E−05 −1.10E−05 9.00E−05 1.61E−04 19.98 1.25E−04 8.10E−05 6.20E−05 2.70E−05 7.70E−05 6.90E−05 4.40E−05 −1.80E−05 1.02E−04 1.56E−04 20.00 1.06E−04 6.20E−05 7.10E−05 2.80E−05 9.10E−05 6.70E−05 5.60E−05 −1.50E−05 1.05E−04 1.71E−04

The advantage of SEC-HPLC analysis was that the insufficiently PEGylated batches, e.g. containing less than 5 PEGylated sites per htCBS C15S protein and/or containing any unmodified htCBS C15S, were characterized by longer retention time than the desired ones and were swiftly identified for rescue by adding more PEG molecules to the reaction mixture (in-process monitoring of PEGylation). The unmodified htCBS C15S sample was observed to have a retention time of 11.97 minutes. The sample from the 6-70 and 8-23 batches were observed to have a retention time of 9.67 minutes. The sample from the 6-89 batch was observed to have a retention time of 9.88 minutes. The sample from the 8-22 batch was observed to have a retention time of 9.95 minutes. Therefore, a retention time of greater than 9.67 minutes was determined to indicate insufficient PEGylation.

The sample from the 8-14 batch was observed to have a retention time of 9.57 minutes. The sample from the 8-15 batch was observed to have a retention time of 9.53 minutes. The sample from the 8-16 batch was observed to have a retention time of 9.55 minutes. The sample from the 8-24 batch was observed to have a retention time of 9.55 minutes. The sample from the 8-25 batch was observed to have a retention time of 9.58 minutes. Therefore, a retention time of less than 9.53 minutes was determined to indicate acceptable PEGylation.

Another method was based on non-reduced capillary electrophoresis allowing resolution and quantification of multiple 20NHS PEG-htCBS C15S species (FIG. 1). Insufficient PEGylation of the batch was characterized by the presence of unmodified enzyme and an increased occurrence of low molecular weight 20NHS PEG-htCBS C15S species. Therefore, this method was found suitable to define strict acceptance criteria for the 20NHS PEG-htCBS C15S final product.

The protein sequence of htCBS C15S contains 30 lysine residues, which all represent potential sites for NHS ester PEGylation. To identify the lysine residues involved in PEGylation, LC-UV-MS was performed three 20NHS PEG-htCBS C15S batches. After de-PEGylation, the formerly PEGylated lysines were left with a linker that enabled their differentiation from unmodified lysines and quantification via peptide mapping after cleavage with Asp-N endopeptidase and comparison to similarly processed unmodified htCBS C15S. LC-MS analysis was performed after Asp-N digest of unmodified htCBS C15S as a reference protein (control).

Table 2 shows the relative abundance of peptides and was used to calculate the number of PEGs per peptide for the reference enzyme, an alkylated, unmodified htCBS C15S, identified by LC/MS/MS. (RT=retention time).

TABLE 2 NHS ester PEGylation mapping of a reference enzyme Sequence SEQ ID Theoretical Observed Δ mass RT Location Sequence NO mass (Da) Mass (Da) (ppm) (min) Abundance   2-34 PSETPQAEVGPTGSPHRSGPHSAKGSLE  2 3294.59 3294.59  0.64 33.8 462828 KGSPE   9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE  3 2584.26 2584.26  0.07 30.8 449108  35-46 DKEAKEPLWIRP  4 1480.80 1480.80 -0.08 50.6 323088  40-46 EPLWIRP  5  909.51  909.51  0.71 54.5 1369777  47-78 DAPSRCTWQLGRPASESPHHHTAPAKS  6 3528.78 3528.78  0.13 49 1805933 PKILP  79-85 DILKKIG  7  785.50  785.50  0.06 41.9 1034520  86-106 DTPMVRINKIGKKFGLKCELL  8 2459.38 2459.38  0.07 59.8 752660 107-119 AKCEFFNAGGSVK  9 1413.67 1413.67  0.28 41.5 865418 120-128 DRISLRMIE 10 1131.61 1131.61  0.74 51.3 1259968 120-128 DRISLRMIE 10 1147.60 1147.60  0.34 43.8 42144 133-139 DGTLKPG 11  686.36  686.36  0.72 24 665344 140-178 DTIIEPTSGNTGIGLALAAAVRGYRCIIV 12 4147.15 4147.15 -0.29 92.2 422022 MPEKMSSEKV 179-197 DVLRALGAEIVRTPTNARF 13 2098.16 2098.17  0.21 64.2 3651887 179-197 DVLRALGAEIVRTPTNARF 13 2099.15 2099.15  1.17 63.7 1401992 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2597.34 2597.33 -0.2 58.1 1160943 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2598.32 2598.32  0.17 58.8 543933 221-233 DQYRNASNPLAHY 15 1547.71 1547.71  0.22 40.9 209797 221-233 DQYRNASNPLAHY 15 1548.70 1548.70  1.59 40.3 156242 221-233 DQYRNASNPLAHY 15 1548.70 1548.70  0.79 41.2 708853 238-244 DEILQQC 16  904.40  904.40  0.12 33.1 258834 245-248 DGKL 17  431.24  431.24  0.01 19.7 98742 249-269 DMLVASVGTGGTITGIARKLK 18 2087.18 2087.18  0.04 59.3 458054 249-269 DMLVASVGTGGTITGIARKLK 18 2103.17 2103.17  0.42 55.3 112910 270-301 EKCPGCRIIGVDPEGSILAEPEELNQTEQ 19 3632.70 3632.70 -0.18 57.7 99941 TTY 302-308 EVEGIGY 20  765.35  765.36  0.99 38.3 356953 309-315 DFIPTVL 21  803.44  803.44 -0.02 66.5 1980222 316-320 DRTVV 22  588.32  588.32  0.75 27.3 891244 321-327 DKWFKSN 23  923.45  923.45  0.03 35.3 209620 328-365 DEEAFTFARMLIAQEGLLCGGSAGSTV 24 3937.96 3937.95 -0.26 95.1 200461 AVAVKAAQELQ 333-348 TFARMLIAQEGL 25 1735.88 1735.87 -1.79 47.2 212890 363-375 ELQEGQRCVVILP 26 1539.81 1539.81  0.31 55.7 81479 366-375 EGQRCVVILP 27 1169.62 1169.62 -0.36 53.8 375007 376-387 DSVRNYMTKFLS 28 1459.71 1459.71 -0.15 55.3 1194612 388-400 DRWMLQKGFLKEE 29 1678.85 1678.85  0.44 52.2 836829 388-400 DRWMLQKGFLKEE 29 1694.85 1694.85  0.39 49.4 129978 401-413 DLTEKKPWWWHLR 30 1793.94 1793.94  0.19 56.7 2238451

Theoretical molecular masses (mono-isotopic) were calculated using MassHunter BioConfirm software (Agilent, Santa Clara, Calif.). The observed masses (mono-isotopic) were within 5 ppm of their theoretical value. Abundances were determined using MassHunter software (Agilent, Santa Clara, Calif.). Peptide 120-128 was observed to have the modification of a 1*oxidation. Peptide 179-197 was observed to have the modification of a 1*deamidation. Peptide 198-220 was observed to have the modification of a 1*deamidation. Peptide 221-233 was observed to have the modification of a 1*deamidation. Peptide 221-233 was observed to have the modification of a 1*deamidation, 249-269 was observed to have the modification of a 1*oxidation. Peptide 388-400 was observed to have the modification of a 1*oxidation.

LC-MS analysis was performed after Asp-N digest of reduced, alkylated, PEGylated 20NHS PEG-htCBS C15S (batch #1). Table 3 shows the relative abundance of peptides and was used to calculate the number of PEGs per peptide for an Asp-N digest of reduced, alkylated, PEGylated 20NHS PEG-htCBS C15S (batch #1), identified by LC/MS/MS.

TABLE 3 NHS ester PEGylation mapping of Asp-N digest of reduced, alkylated, PEGylated 20NHS PEG-htCBS C15S (batch #1) Sequence SEQ ID Theoretical Observed Δ mass RT Location Sequence NO mass (Da) Mass (Da) (ppm) (min) Abundance   2-34 PSETPQAEVGPTGSPHRSGPHSAKGS  2 3294.59 3294.59 -0.46 33.8 74988 LEKGSPE   2-34 PSETPQAEVGPTGSPHRSGPHSAKGS  2 3408.62 3408.62  1.2 36.7 285758 LEKGSPE   2-34 PSETPQAEVGPTGSPHRSGPHSAKGS  2 3522.65 3522.65  0.51 39.3 251006 LEKGSPE   9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE  3 2584.26 2584.26  0.43 30.8 50722   9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE  3 2698.29 2698.3  1.34 34.6 156217   9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE  3 2812.33 2812.33  0.43 37.6 59784  35-46 DKEAKEPLWIRP  4 1480.8 1480.8  0.14 50.5 419366  35-46 DKEAKEPLWIRP  4 1594.84 1594.83 -0.42 53.3 115586  40-46 EPLWIRP  5  909.51  909.51  0.36 54.4 1267685  47-78 DAPSRCTWQLGRPASESPHHHTAPA  6 3528.78 3528.78  0.35 49 916229 KSPKILP  47-78 DAPSRCTWQLGRPASESPHHHTAPA  6 3642.81 3642.81  0.3 51.2 536395 KSPKILP  79-85 DILKKIG  7  785.5  785.5  0.2 41.9 1447731  79-85 DILKKIG  7  899.53  899.53  0.01 47.1 49654  86-106 DTPMVRINKIGKKFGLKCELL  8 2459.38 2459.38  0 59.8 278431  86-106 DTPMVRINKIGKKFGLKCELL  8 2573.41 2573.41  0.2 63.4 207777  86-106 DTPMVRINKIGKKFGLKCELL  8 2573.41 2573.41  0.39 66 268441 107-119 AKCEFFNAGGSVK  9 1413.67 1413.67  0.21 41.5 1031048 120-128 DRISLRMIE 10 1131.61 1131.61  0.63 51.2 1738225 120-128 DRISLRMIE 10 1147.6 1147.6 -0.19 43.8 74835 133-139 DGTLKPG 11  686.36  686.36  0.53 24 837583 140-178 DTIIEPTSGNTGIGLALAAAVRGYRCI 12 4147.15 4147.15 -0.03 92.2 575055 IVMPKMSSEKV 179-197 DVLRALGAEIVRTPTNARF 13 2098.16 2098.17  0.18 64.1 4896736 179-197 DVLRALGAEIVRTPTNARF 13 2099.15 2099.15  0.59 63.6 1870150 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2597.34 2597.34  0.11 58 1302818 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2598.32 2598.32  0.17 58.7 591137 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2711.37 2711.37  1 63.5 65301 221-233 DQYRNASNPLAHY 15 1547.71 1547.71 -0.06 40.9 256919 221-233 DQYRNASNPLAHY 15 1548.7 1548.7  1.11 40.3 198652 221-233 DQYRNASNPLAHY 15 1548.7 1548.7  0.61 41.2 802841 238-244 DEILQQC 16  904.4  904.4 -0.35 33.1 216368 245-248 DGKL 17  431.24  431.24  0.15 19.9 75839 245-248 DGKL 17  545.27  545.27 -0.29 31.7 73171 249-269 DMLVASVGTGGTITGIARKLK 18 2087.18 2087.18 -0.18 59.2 458170 249-269 DMLVASVGTGGTITGIARKLK 18 2103.17 2103.17  0.16 55.2 130431 270-301 EKCPGCRIIGVDPEGSILAEPEELNQT 19 3632.7 3632.7  1.24 57.6 37257 EQTTY 270-301 EKCPGCRIIGVDPEGSILAEPEELNQT 19 3746.73 3746.72 -1.28 59.5 23764 EQTTY 302-308 EVEGIGY 20  765.35  765.35  0.03 38.3 274612 309-315 DFIPTVL 21  803.44  803.44  0.29 66.4 2313422 316-320 DRTVV 22  588.32  588.32  0.11 27.2 1216696 321-327 DKWFKSN 23  923.45  923.45  0.03 35.3 100188 321-327 DKWFKSN 23 1037.48 1037.48 -0.64 41.9 44718 328-365 DEEAFTFARMLIAQEGLLCGGSAGS 24 3937.96 3937.95 -0.4 95.1 82719 TVAVAVKAAQELQ 333-348 TFARMLIAQEGL 25 1735.88 1735.87 -1.84 47.2 158013 363-375 ELQEGQRCVVILP 26 1539.81 1539.81  0.05 55.6 109572 366-375 EGQRCVVILP 27 1169.62 1169.62  0.17 53.7 425023 376-387 DSVRNYMTKFLS 28 1459.71 1459.71  0.34 55.2 1488072 388-400 DRWMLQKGFLKEE 29 1678.85 1678.85 -0.04 52.1 1018180 388-400 DRWMLQKGFLKEE 29 1694.85 1694.85 -0.05 49.2 138406 388-400 DRWMLQKGFLKEE 29 1792.88 1792.88 -0.32 57.4 61595 401-413 DLTEKKPWWWHLR 30 1793.94 1793.94  0.13 56.7 971171 401-413 DLTEKKPWWWHLR 30 1907.97 1907.97  0.08 60.8 1558334 401-413 DLTEKKPWWWHLR 30 2022 2022  0.4 66.5 242737

Further, 5 peptides with 2 lysines and 1 peptide with 4 lysines were identified, of which one lysine residue was found inconsistently PEGylated: K36/39, K72/75, K82/83, K94/97/98/102, K322/325 and K394/398. The estimated number of PEG/peptide was calculated using the following formula: Estimated (PEG/peptide) ratio=(Abundance(1*Linker)+2*Abundance(2*Linker))/((Abundance(No Linker)+Abundance(1*Linker)+Abundance(2*Linker)). Since PEGs are known in the art to not migrate according to their molecular weight in gels or chromatographic applications due to a significant solvation envelope and thus increased hydrodynamic radius, the extent of PEGylation was estimated to be in the range of 5.0±0.5 PEGs per CBS monomer. Specifically, the number PEG molecules per peptide was estimated to be 1.29 for 2-34, 1.30 for 9-34, 0.22 for 35-46, 0.37 for 47-78, 0.03 for 79-85 and 198-220, 0.63 for 86-106, 0.49 for 245-248, 0.39 for 270-301, 0.31 for 321-327, and 0.05 for 388-400.

Peptide 2-34 was observed to have the modification of a 1*linker. Peptide 2-34 was observed to have the modification of a 2*linker. Peptide 9-34 was observed to have the modification of a 1*linker. Peptide 9-34 was observed to have the modification of a 2*linker. Peptide 35-46 was observed to have the modification of a 1*linker. Peptide 47-78 was observed to have the modification of a 1*linker. Peptide 79-85 was observed to have the modification of a 1*linker. Peptide 86-106 was observed to have the modification of a 1*linker. Peptide 86-106 was observed to have the modification of a 1*linker. Peptide 120-128 was observed to have the modification of a 1*oxidation. Peptide 179-197 was observed to have the modification of a 1*deamidation. Peptide 198-220 was observed to have the modification of a 1*deamidation. Peptide 198-220 was observed to have the modification of a 1*linker. Peptide 221-233 was observed to have the modification of a 1*deamidation. Peptide 221-233 was observed to have the modification of a 1*deamidation. Peptide 245-248 was observed to have the modification of a 1*linker. Peptide 249-269 was observed to have the modification of a 1*oxidation. Peptide 270-301 was observed to have the modification of a 1*linker. Peptide 321-327 was observed to have the modification of a 1*linker. Peptide 388-400 was observed to have the modification of a 1*oxidation. Peptide 388-400 was observed to have the modification of a 1*linker. Peptide 401-413 was observed to have the modification ofa 1*linker. Peptide 401-413 was observed to have the modification of a 2*linker.

LC-MS analysis was performed after Asp-N digest of reduced, alkylated, PEGylated 20NHS PEG-htCBS C15S (batch #2). Table 4 shows the relative abundance of peptides and estimated number of PEGs per peptide for an Asp-N digest of a reduced, alkylated, PEGylated 20NHS PEG-htCBS C15S (batch #2), identified by LC/MS/MS.

TABLE 4 NHS ester PEGylation mapping of Asp-N digest of reduced, alkylated, PEGylated 20NHS PEG-htCBS C15S (batch #2) Sequence SEQ ID Theoretical Observed Δ mass RT Location Sequence NO mass (Da) Mass (Da) (ppm) (min) Abundance   2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL  2 3294.5865 3294.5858 -0.21 34.1 91654 EKGSPE   2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL  2 3408.6182 3408.6173 -0.26 37.1 212552 EKGSPE   2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL  2 3522.6499 3522.6505  0.17 39.6 116731 EKGSPE   9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE  3 2584.263 2584.2621 -0.35 31.2 110005   9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE  3 2698.2947 2698.2944 -0.1 35 207053   9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE  3 2812.3264 2812.3283  0.68 37.6 50468  35-46 DKEAKEPLWIRP  4 1480.8038 1480.804  0.15 51.1 130726  35-46 DKEAKEPLWIRP  4 1594.8355 1594.836  0.31 53.8 26208  40-46 EPLWIRP  5  909.5072  909.5075  0.31 54.9 370392  47-78 DAPSRCTWQLGRPASESPHHHTAPAK  6 3528.7797 3528.7769 -0.79 49.4 608763 SPKILP  47-78 DAPSRCTWQLGRPASESPHHHTAPAK  6 3642.8114 3642.8084 -0.82 51.6 211918 SPKILP  79-85 DILKKIG  7  785.5011  785.5017  0.78 42.3 454812  79-85 DILKKIG  7  899.5328  899.5338  1.11 47.5 11289  86-106 DTPMVRINKIGKKFGLKCELL  8 2459.3756 2459.3774  0.73 60.8 179277  86-106 DTPMVRINKIGKKFGLKCELL  8 2573.4073 2573.4077  0.16 64.6 92660  86-106 DTPMVRINKIGKKFGLKCELL  8 2573.4073 2573.4068 -0.19 67.3 117060 107-119 AKCEFFNAGGSVK  9 1413.6711 1413.6716  0.35 42 495351 120-128 DRISLRMIE 10 1131.607 1131.6075  0.46 51.7 664834 120-128 DRISLRMIE 10 1147.6019 1147.6023  0.35 44.3 33046 133-139 DGTLKPG 11  686.3599  686.3603  0.61 24.3 285635 140-178 DTIIEPTSGNTGIGLALAAAVRGYRCII 12 4147.148 4147.1451 -0.7 93.5 181549 VMPEKMSSEKV 179-197 DVLRALGAEIVRTPTNARF 13 2098.1647 2098.1657  0.49 65.3 2248724 179-197 DVLRALGAEIVRTPTNARF 13 2099.1487 2099.1508  0.99 64.8 936714 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2597.335 2597.3345 -0.23 58.7 635630 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2598.3191 2598.3202  0.45 59.5 352959 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2711.3667 2711.3649 -0.66 64.5 23552 221-233 DQYRNASNPLAHY 15 1547.7117 1547.7118  0.06 41.4 128480 221-233 DQYRNASNPLAHY 15 1548.6957 1548.6971  0.9 40.8 111326 221-233 DQYRNASNPLAHY 15 1548.6957 1548.6955 -0.17 41.7 405793 238-244 DEILQQC 16  904.396  904.3962  0.23 33.5 126894 245-248 DGKL 17  431.238  431.2381  0.12 20.5 40770 245-248 DGKL 17  545.2697  545.2699  0.37 32.2 26289 249-269 DMLVASVGTGGTITGIARKLK 18 2087.1773 2087.178  0.34 59.8 301153 249-269 DMLVASVGTGGTITGIARKLK 18 2103.1722 2103.174  0.86 55.3 81381 270-301 EKCPGCRIIGVDPEGSILAEPEELNQTE 19 3632.6975 3632.6996  0.58 58.1 51146 QTTY 270-301 EKCPGCRIIGVDPEGSILAEPEELNQTE 19 3746.7292 3746.7269 -0.61 60 18541 QTTY 302-308 EVEGIGY 20  765.3545  765.3549  0.49 38.7 244178 309-315 DFIPTVL 21  803.4429  803.4436  0.86 66.9 1102772 316-320 DRTVV 22  588.3231  588.3235  0.59 27.7 482942 321-327 DKWFKSN 23  923.4501  923.4504  0.33 35.9 41312 321-327 DKWFKSN 23 1037.4818 1037.4826  0.77 42.5 10692 328-365 DEEAFTFARMLIAQEGLLCGGSAGST 24 3937.9554 3937.9519 -0.89 95.6 35286 VAVAVKAAQELQ 333-348 TFARMLIAQEGL 25 1735.875 1735.8712 -2.19 47.7 113391 363-375 ELQEGQRCVVILP 26 1539.8079 1539.8083  0.26 55.9 44354 366-375 EGQRCVVILP 27 1169.6227 1169.6238  0.95 54.1 143135 376-387 DSVRNYMTKFLS 28 1459.713 1459.7144  0.96 56.1 679328 388-400 DRWMLQKGFLKEE 29 1678.8501 1678.8505  0.22 52.8 428907 388-400 DRWMLQKGFLKEE 29 1694.845 1694.8454  0.24 69.9 80607 388-400 DRWMLQKGFLKEE 29 1792.8818 1792.881 -0.45 58.2 20614 401-413 DLTEKKPWWWHLR 30 1793.9366 1793.9372  0.38 57.6 638701 401-413 DLTEKKPWWWHLR 30 1907.9683 1907.9691  0.45 62.1 633624 401-413 DLTEKKPWWWHLR 30 2022 2022.001  0.49 67.8 84315

The number PEG molecules per peptide was estimated to be 1.06 for 2-24, 0.84 for 9-34, 0.17 for 35-46, 0.26 for 47-78, 0.02 for 79-85 and 198-220, 0.54 for 86-106, 0.39 for 245-248, 0.27 for 270-301, 0.21 for 321-327, 0.04 for 388-400, and 0.59 for 401-413. Peptide 2-34 was observed to have the modification of a 1*linker. Peptide 2-34 was observed to have the modification of a 2*linker. Peptide 9-34 was observed to have the modification of a 1*linker. Peptide 9-34 was observed to have the modification of a 2*linker. Peptide 35-46 was observed to have the modification of a 1*linker. Peptide 47-78 was observed to have the modification of a 1*linker. Peptide 79-85 was observed to have the modification of a 1*linker. Peptide 86-106 was observed to have the modification of a 1*linker. Peptide 86-106 was observed to have the modification of a 1*linker. Peptide 120-128 was observed to have the modification of a 1*oxidation. Peptide 179-197 was observed to have the modification of a 1*deamidation. Peptide 198-220 was observed to have the modification of a 1*deamidation. Peptide 198-220 was observed to have the modification of a 1*linker. Peptide 221-233 was observed to have the modification of a 1*deamidation. Peptide 221-233 was observed to have the modification of a 1*deamidation. Peptide 245-248 was observed to have the modification of a 1*linker. Peptide 249-269 was observed to have the modification of a 1*oxidation. Peptide 270-301 was observed to have the modification of a 1*linker. Peptide 321-327 was observed to have the modification of a 1*linker. Peptide 388-400 was observed to have the modification of a 1*oxidation. Peptide 388-400 was observed to have the modification of a 1*linker. Peptide 401-413 was observed to have the modification of a 1*linker. Peptide 401-413 was observed to have the modification of a 2*linker.

LC-MS analysis was performed after Asp-N digest of reduced, alkylated, PEGylated 20NHS PEG-htCBS C15S (batch #3). Table 5 shows the relative abundance of peptides and estimated number of PEGs per peptide identified by LC/MS/MS for batch #3.

TABLE 5 NHS ester PEGylation mapping of Asp-N digest of reduced, alkylated, PEGylated 20NHS PEG-htCBS C15S (batch #3) Sequence SEQ ID Theoretical Observed Δ mass RT Location Sequence NO mass (Da) Mass (Da) (ppm) (min) Abundance   2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL  2 3294.5865 3294.5829 -1.09 34.3 60072 EKGSPE   2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL  2 3408.6182 3408.6154 -0.82 37.2 199290 EKGSPE   2-34 PSETPQAEVGPTGSPHRSGPHSAKGSL  2 3522.6499 3522.6508  0.26 39.7 159538 EKGSPE   9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE  3 2584.263 2584.2638  0.31 31.3 105797   9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE  3 2698.2947 2698.294 -0.25 35.2 261221   9-34 EVGPTGSPHRSGPHSAKGSLEKGSPE  3 2812.3264 2812.3259 -0.18 37.6 96655  35-46 DKEAKEPLWIRP  4 1480.8038 1480.8039  0.09 51.3 86032  35-46 DKEAKEPLWIRP  4 1594.8355 1594.8362  0.44 54 18790  40-46 EPLWIRP  5  909.5072  909.5078  0.62 55 395424  47-78 DAPSRCTWQLGRPASESPHHHTAPAK  6 3528.7797 3528.78  0.08 49.6 595498 SPKILP  47-78 DAPSRCTWQLGRPASESPHHHTAPAK  6 3642.8114 3642.8084 -0.81 51.7 272886 SPKILP  79-85 DILKKIG  7  785.5011  785.5017  0.81 42.5 446682  79-85 DILKKIG  7  899.5328  899.5347  2.11 47.7 12609  86-106 DTPMVRINKIGKKFGLKCELL  8 2459.3756 2459.3745 -0.45 60.7 145186  86-106 DTPMVRINKIGKKFGLKCELL  8 2573.4073 2573.4088  0.58 64.5 101537  86-106 DTPMVRINKIGKKFGLKCELL  8 2573.4073 2573.4057 -0.62 67.3 126416 107-119 AKCEFFNAGGSVK  9 1413.6711 1413.6721  0.71 42.2 506761 120-128 DRISLRMIE 10 1131.607 1131.6075  0.42 51.9 725914 120-128 DRISLRMIE 10 1147.6019 1147.602  0.04 44.3 29014 133-139 DGTLKPG 11  686.3599  686.3601  0.23 24.5 273520 140-178 DTIIEPTSGNTGIGLALAAAVRGYRCII 12 4147.148 4147.1398 -1.97 93.4 188751 VMPEKMSSEKV 179-197 DVLRALGAEIVRTPTNARF 13 2098.1647 2098.1663  0.73 65.2 2212569 179-197 DVLRALGAEIVRTPTNARF 13 2099.1487 2099.1511  1.12 64.7 930809 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2597.335 2597.3339 -0.44 58.7 645205 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2598.3191 2598.3196  0.2 59.5 348068 198-220 DSPESHVGVAWRLKNEIPNSHIL 14 2711.3667 2711.3668  0.04 64.4 31254 221-233 DQYRNASNPLAHY 15 1547.7117 1547.7124  0.48 41.7 119215 221-233 DQYRNASNPLAHY 15 1548.6957 1548.6981  1.56 41.1 113439 221-233 DQYRNASNPLAHY 15 1548.6957 1548.6959  0.14 42 404271 238-244 DEILQQC 16  904.396  904.3962  0.16 33.7 137033 245-248 DGKL 17  431.238  431.2384  0.93 20.8 41279 245-248 DGKL 17 545.2697  545.2699  0.44 32.4 38708 249-269 DMLVASVGTGGTITGIARKLK 18 2087.1773 2087.1766 -0.32 59.8 378816 249-269 DMLVASVGTGGTITGIARKLK 18 2103.1722 2103.1743  1 55.3 81123 270-301 EKCPGCRIIGVDPEGSILAEPEELNQTE 19 3632.6975 3632.6934 -1.13 58.1 69301 QTTY 270-301 EKCPGCRIIGVDPEGSILAEPEELNQTE 19 3746.7292 3746.7248 -1.17 60 28571 QTTY 302-308 EVEGIGY 20  765.3545  765.3551  0.76 39 267474 309-315 DFIPTVL 21  803.4429  803.4434  0.66 66.9 1030604 316-320 DRTVV 22  588.3231  588.3236  0.72 27.9 474399 321-327 DKWFKSN 23  923.4501  923.4498 -0.3 36.3 46084 321-327 DKWFKSN 23 1037.4818 1037.4838  1.93 42.8 12212 328-365 DEEAFTFARMLIAQEGLLCGGSAGST 24 3937.9554 3937.9541 -0.33 95.6 45366 VAVAVKAAQELQ 333-348 TFARMLIAQEGL 25 1735.875 1735.87 -2.88 48 99444 363-375 ELQEGQRCVVILP 26 1539.8079 1539.8083  0.26 56 45283 366-375 EGQRCVVILP 27 1169.6227 1169.6233  0.52 54.1 162480 376-387 DSVRNYMTKFLS 28 1459.713 1459.7127 -0.16 56.2 715580 388-400 DRWMLQKGFLKEE 29 1678.8501 1678.8503  0.08 53 430875 388-400 DRWMLQKGFLKEE 29 1694.845 1694.8452  0.12 50.2 71467 388-400 DRWMLQKGFLKEE 29 1792.8818 1792.8825  0.39 58.2 24688 401-413 DLTEKKPWWWHLR 30 1793.9366 1793.9372  0.38 57.7 524828 401-413 DLTEKKPWWWHLR 30 1907.9683 1907.969  0.38 62 701248 401-413 DLTEKKPWWWHLR 30 2022 2022.0014  0.69 67.6 130215

The number PEG molecules per peptide was estimated to be 1.24 for 2-34, 0.98 for 9-34, 0.18 for 35-46, 0.31 for 47-78, 0.03 for 79-85 and 198-220, 0.61 for 86-106, 0.48 for 245-248, 0.29 for 270-301, 0.21 for 321-327, 0.05 for 388-400, and 0.71 for 401-413.

Peptide 2-34 was observed to have the modification of a 1*Linker. Peptide 2-34 was observed to have the modification of a 2*Linker. Peptide 9-34 was observed to have the modification of a 1*Linker. Peptide 9-34 was observed to have the modification of a 2*Linker. Peptide 35-46 was observed to have the modification of a 1*Linker. Peptide 47-78 was observed to have the modification of a 1*Linker. Peptide 79-85 was observed to have the modification of a 1*Linker. Peptide 86-106 was observed to have the modification of a 1*linker. Peptide 86-106 was observed to have the modification of a 1*linker. Peptide 120-128 was observed to have the modification of a 1*oxidation. Peptide 179-197 was observed to have the modification of a 1*deamidation. Peptide 198-220 was observed to have the modification of a 1*deamidation. Peptide 198-220 was observed to have the modification of a 1*linker. Peptide 221-233 was observed to have the modification of a 1*deamidation. Peptide 221-233 was observed to have the modification of a 1*deamidation. Peptide 245-248 was observed to have the modification of a 1*linker. Peptide 249-269 was observed to have the modification of a 1*oxidation. Peptide 270-301 was observed to have the modification of a 1*linker. Peptide 321-327 was observed to have the modification of a 1*linker. Peptide 388-400 was observed to have the modification of a 1*oxidation. Peptide 388-400 was observed to have the modification of a 1*linker. Peptide 401-413 was observed to have the modification of a 1*linker. Peptide 401-413 was observed to have the modification of a 2*linker.

Seven lysines (K25, K30, K211, K247, K271, K405 and K406) were unambiguously identified as being PEGylated to a certain extent, out of which K25 and K30 residues seemed the only ones to be always modified to the full extent among the 3 batches.

Compared to all the previous PEG-htCBS C15S conjugates, the 20NHS PEG-htCBS C15S showed increased viscosity (data not shown) and thus there was a concern of sufficient free, unreacted PEG removal during the final (post PEGylation) buffer exchange. Indeed, processing of the initial batches (for example LAB and 10L) yielded a large amount of unreacted PEG carried over to the final product. Corrective measures, which included removal of 20% DMSO from the mixture, higher MWCO membrane cartridge for TFF unit (100 versus 30 kDa), 3-fold larger surface area and 2-fold dilution of the PEGylation mixture prior buffer exchange, all helped to reduce the viscosity and assisted in better clearance of free PEG during the final formulation (TR1 and TR2).

Example 5. Attenuation of 200MA PEG-htCBS C15S Efficacy in HO Mice

Repeated administration in HO mice was defined as subcutaneous injections of 7.5 mg/kg 200MA PEG-htCBS C15S for 5 consecutive days during the first dose-week. Dosing was interrupted for a period of 10 days and restarted again for an additional 5 consecutive injections in the second dose-week. Plasma samples were collected before the very first dose for baseline values, 24 hours after the 2nd, 4th, and 5th injections in both dose-weeks to track the enzyme's efficacy and during washout periods to follow recovery of the baseline values (a week after the last dose in both dose-weeks, prior to the first dose of the dose-week 2 and two weeks after the very last dose). The study showed in an apparent attenuation of 200MA PEG-htCBS C15S efficacy in the dose-week 2 compared to dose-week 1 (see, Table 7).

A bulkier maleimide PEG molecule did not resolve the attenuation of PEG-htCBS C15S efficacy in washout experiments. Since the use of maleimide PEG, which specifically targets available sulfhydryl residues, was known to yield a well-defined species, the initial approach to reduce the loss of 200MA PEG-htCBS C15S efficacy in washout experiments was to increase the coverage of the htCBS C15S protein with a bulkier maleimide PEG. The previously published data showed that PEGylation of htCBS with larger maleimide PEG molecule did not affect catalytic activity in vitro and that 40 kDa linear (ME-400MA) or branched (GL4-400MA) maleimide PEG molecules had similar pharmacokinetics and pharmacodynamics after a single injection in HO mice compared to 200MA PEG-htCBS C15S. See, Bublil et al., J Clin Invest 2016, 126, (6), 2372-84 and International application PCT/2016/061050, which are hereby incorporated by reference in their entireties. Table 6 shows that the PEGylation of htCBS C15S does not substantially affect the enzyme's catalytic activity regardless of the PEG moiety size and the target group.

TABLE 6 CBS specific activities of unmodified htCBS C15S and PEG-htCBS C15S conjugates Active moiety (target CBS specific activity (U/mg of PEG moiety Size & structure group) protein ± SD) None N/A N/A 1233 ± 201 ME-020MA 2 kDa linear maleimide (—SH) 1184 ± 95  ME-200MA0B 20 kDa linear maleimide (—SH) 1339 ± 226 ME-400MA 40 kDa linear maleimide (—SH) 1242 ± 158 GL4-400MA 40 kDa 4-arm branched maleimide (—SH) 1288 ± 221 GL2-800MA 80 kDa 2-arm branched maleimide (—SH) 1168 ± 134 ME-050GS 5 kDa linear NHS ester (—NH₂) 1250 ± 52  ME-100GS 10 kDa linear NHS ester (—NH₂) 1233 ± 173 ME-200GS 20 kDa linear NHS ester (—NH₂) 1339 ± 242

The dosing regimen was specifically designed to elicit immune response to rank conjugates based on their immunogenic properties. The repeated uninterrupted dosing of 200MA PEG-htCBS C15S to homocystinuric HO mice has previously been shown to result in a retained efficacy and a significant decrease of plasma Hcy levels 24 hours after injection for a period of 2 months, although both the baseline level (72 hours post injection) of plasma Hcy and the peak effect of 200MA PEG-htCBS C15S (24 hours post injection) were decreasing over time. The most reasonable explanation for such attenuation of activity is the host's immune response, which may adversely impact both the efficacy and the safety and is relatively common among approved therapeutic enzymes. See, Baldo et al., BioDrugs: clinical immunotherapeutics, biopharmaceuticals and gene therapy 2015, 29, (1), 31-55, which is hereby incorporated by reference in its entirety.

The immunogenicity may stem from the trace amounts of unmodified enzyme or insufficiently masked htCBS C15S with site selective maleimide PEG molecule conjugation. Therefore, a complete absence of unmodified enzyme and more generous masking of potential immunogenic epitopes of PEG-htCBS C15S conjugate from the immune system by increased PEGylation would be expected to prevent the decrease in efficacy.

Example 6. Retaining Efficacy of 20NHS PEG-htCBS in HO Mice

NHS ester PEG-htCBS C15S conjugates modified with ME-050GS (5NHS PEG-htCBS C15S) and ME-200GS (20NHS PEG-htCBS C15S) were analyzed in washout experiments, and results were compared to results from similar analysis of maleimide PEGylated conjugates. Table 7 provides the percent changes in metabolites resulting from administration of different PEG-htCBS C15S conjugates over 3 weeks. (W1=1 week, W2=2 weeks, and W3=3 weeks)

TABLE 7 Maximal changes in the plasma sulfur amino acid levels after treatment in HO mice with PEGylated htCBS C15S conjugates Hcy decrease at trough (%) Cth increase at peak (%) Cys increase at peak (%) PEG molecule W1 W2 W3 W1 W2 W3 W1 W2 W3 PBS (control) −8.0 −8.1 n/a −1 −19 n/a −18.1 4.7 n/a ME-400MA 73.2 64.9 58.6 383 329 365 17.2 6.3 8.0 GL4-400MA 74.6 41.1 34.0 715 723 449 97.8 47.5 51.8 ME-400MA 74.9 52.0 41.5 696 649 443 71.9 44.3 43.6 ME-050GS 74.8 58.5 42.1 978 795 833 70.7 75.0 58.3 ME-200GS 76.9 70.2 65.8 676 492 432 58.7 60.6 50.8 ME-200MA 68.0 24.0 n/a 796 508 n/a 35.7 16.1 n/a ME-200MA 82.0 56.6 50.4 700 866 335 71.5 37.9 31.5 ME-200MA 80.4 60.6 50.1 1023 764 1100 76.6 77.3 58.3

The 5NHS PEG-htCBS C15S showed an excellent pharmacodynamics response after the first set of injections but a significant loss of efficacy was observed in the subsequent dose-weeks for Hcy. Hcy levels dropped to 48/80/111 μM (baseline levels: 192/175/201 μM) with a simultaneous increase in Cth levels to 43/35/37 μM (baseline levels: 4/4/4 μM) and concurrent increase of Cys levels to 219/225/174 μM (baseline levels: 129/137/146 μM) in dose-weeks 1/2/3 (FIG. 6 and Table 7).

20NHS PEG-CBS showed a balanced response in all three dose-weeks: the plasma Hcy concentration dropped to 38/49/56 μM from baseline levels 164/165/143 μM with a subsequent increase in Cth levels to 40/30/27 μM (baseline levels: 5/4/4 μM) and concurrent increase of Cys levels to 234/237/222 μM (baseline levels: 147/149/148 μM) in dose-weeks 1/2/3. As a control experiment, two additional batches of 200MA PEG-htCBS C15S were analyzed in a washout experiment and attenuated efficacy in subsequent dose-weeks was again observed: plasma Hcy dropped to 44/88/112 μM (baseline levels: 224/215/224 μM) with consequent increase in Cth levels to 40/31/43 μM (baseline levels: 4/3/4 μM) and concurrent increase of Cys levels to 219/219/196 μM (baseline levels: 124/118/144 μM) in dose-weeks 1/2/3. The 20NHS PEG-CBS showed the least attenuation of the pharmacodynamic effect on plasma metabolites after repeated administration of a PEGylated enzyme to HO mice (Table 7).

Example 7. Rescuing the Phenotype of KO Mice by Treatment with 20NHS CBS-PEG

To assess the potentially beneficial effects of 20NHS PEG-htCBS C15S treatment on overall health and thriving of KO mice.

The body weights and weight gains of 20NHS PEG-htCBS C15S-treated mice (n-16M+11F) were determined every other day from weaning (day 21) up to 35 days of age and compared to sex- and age-matched PBS-treated heterozygous+/−mice (n-14M+13F). Mice received subcutaneous injections 3× a week of 7.5 mg/kg from day 2 of age. Body weight (FIG. 7A) and weight gain (FIG. 7B) of 20NHS PEG-htCBS C15S-treated CBS KO male (gray solid line & circles) and female (gray dashed line & circles) mice are compared to PBS-injected+/−males (black solid line & squares) and females (black dashed line & squares).

At the time of weaning (day 21), no significant differences were found in body weights between both heterozygous or KO mice and males or females receiving either PBS or 20NHS PEG-htCBS C15S except for males versus females on 20NHS PEG-htCBS C15S injections (p<0.01). Two days later (day 23) no significant difference in body weights among study mice was found. From day 25 on, the +/−males gained weight significantly (p<0.001) more and thus increase their body weight more than the +/−females as well as the 20NHS PEG-htCBS C15S-treated KO males or females. In KO mice, weaning led to a brief period of slower growth (days 23 and 25) characterized by a smaller weight gain and practically maintained body weight followed by steady gains in weight. Weight gains of KO males and females treated with 20NHS PEG-htCBS C15S were essentially identical and at days 33 and 35 their weight gains and body weights of treated KO females caught up with those of PBS-injected heterozygous females, while KO males fell behind a bit their heterozygous counterparts. Taken together, 20NHS PEG-htCBS C15S-treated KO mice gained weight steadily with females more than males catching up with the PBS-treated heterozygous mice in terms of body weight at the end of the 5-week treatment period.

Example 8. Sustaining Improved Plasma Levels by Treatment with 20NHS PEG-htCBS C15S

Clinical symptoms of the I278T mice, such as facial alopecia or osteoporosis, take time to develop as well as get corrected with treatment. In this Example, treatment of I278T mice with 20NHS PEG-htCBS C15S was shown to significantly improve or normalize the underlying metabolic imbalance and sustain it over a longer period. FIG. 8A and FIG. 8B show sustained efficacy of 20NHS PEG-htCBS C15S on plasma sulfur amino acids in the I278T mouse model. FIG. 8A showed a sustained improvement in plasma Hcy, Cth, and Cys levels in asymptomatic weaned I278T mice of 3 weeks of age (n=3) treated since then with three weekly SC injections (7.5 mg/kg) for a period of about 9 months. The initial levels recorded prior the first dose at day 21 showed highly elevated Hcy (288 μM), roughly half of the normal Cys levels (129 μM) and low plasma Cth (1.4 μM). Plasma metabolites determined 72 hours after the 3rd (day 28) and the 18th dose (day 63), respectively, illustrated that the lack of dosing during weekends resulted in somewhat a similar profile compared to the initial levels except for Cth, a marker of 20NHS PEG-htCBS C15S activity, for which elevated plasma concentration persisted most likely due to its slow renal clearance.

On the contrary, plasma levels determined 24 hours after 20NHS PEG-htCBS C15S administration (e.g. at day 64 and applies for all the subsequent time points) demonstrate substantially and significantly decreased Hcy levels (105 μM, p<0.001), normalized plasma Cys (209 μM, p<0.001) and elevated Cth (43 μM, p<0.001) compared to the initial values. Similarly, FIG. 8B exemplifies a persistent correction of plasma sulfur amino acids in 26 weeks old 1278T mice (n=10) showing multiple clinical signs of the diseases and treated since then (3× a week, SC, 7.5 mg/kg) for a period of about 6 months. The initial levels recorded prior the first dose at day 182 showed highly elevated Hcy (321 μM), roughly half of the normal Cys levels (127 μM) and low plasma Cth (0.3 μM). The 24 hours after initial administration of 20NHS PEG-htCBS C15S (day 184) resulted in a marked and significant decrease of plasma Hcy (143 μM, p<0.001), normalization of plasma Cys (246 μM, p<0.001) and elevation of Cth (54 μM, p<0.001) compared to the pre-treatment values. The improved plasma metabolite profile was maintained with regular ERT administration for a period of about 6 months. Taken together, regular SC injections of 20NHS PEG-htCBS C15S yielded a sustained improved plasma metabolite profile in the 1278T mouse, thus permitting efficacy studies requiring long-term administration.

Example 9. Normalizing Tissue Metabolite Levels and Improving the Balance of Metabolites

Administration of 20NHS PEG-htCBS C15S was observed to result in correction of tissue metabolites, which correlated to improved plasma metabolites balance in adult 1278T mice. Concentrations of sulfur-containing metabolites in liver, kidney and brain homogenates and corresponding plasma levels of about 2-month-old 1278T mice (n=3) treated with 20NHS PEG-htCBS C15S (SC, 7.5 mg/kg) for a period of 3 weeks compared to age-matched healthy heterozygous mice (n=3) as well as untreated 1278T mice (n=3) were measured. The sulfur-containing metabolites included homocysteine (Hcy), cysteine (Cys), cystathionine (Cth), homolanthionine (Hlth), and lanthionine (Lth). Total homocysteine and cysteine was determined in plasma, while nonprotein-bound fractions of these thiols were measured in tissue homogenates. The 1278T adolescent mice (n=3) were treated for a period of 3 weeks with 20NHS PEG-htCBS C15S (3× a week, SC, 7.5 mg/kg) and compared to age-matched untreated 1278T mice (n=3) and untreated healthy heterozygous controls (n=3). Data were compared using ANOVA followed by Tukey's post hoc test to determine significance: * p<0.05, ** p<0.01, *** p<0.001, ns—non-significant. Results are described by μM in nmol/g of tissue or nM in pmol/g of tissue.

Total Hcy and non-protein bound Hcy was greatly elevated in plasma and liver/kidney/brain homogenates, respectively, in the untreated 1278T mice (310 μM in plasma and 29/21/12 μM in nmols/g of liver/kidney/brain) compared to both heterozygous controls (12 μM in plasma and 8/6/4 μM in nmols/g of liver/kidney/brain) and 20NHS PEG-htCBS C15S-treated 1278T mice (54 μM in plasma and 8/4/1 μM in nmols/g of liver/kidney/brain, p<0.001 for plasma and <0.01 for tissues). The 4.5-fold elevated total Hcy in plasma, non-protein bound Hcy in tissues of the treated 1278T mice was not significantly different from the levels in healthy heterozygous mice.

Total Cys was substantially decreased in plasma (91 μM), but the non-protein bound Cys in liver/kidney/brain homogenates of the untreated 1278T mice (21/432/44 μM in nmols/g of liver/kidney/brain) was not observed to be different compared to the 20NHS PEG-htCBS C15S-treated 1278T mice (224 μM in plasma and 36/265/51 μM in nmols/g of liver/kidney/brain, p<0.01 for plasma and non-significant for tissues). The Cys levels in the treated 1278T mice were similar to those of healthy heterozygous controls (255 μM in plasma and 81/322/44 μM in nmols/g of liver/kidney/brain, p=non-significant).

As anticipated, Cth levels in both plasma and tissues of untreated 1278T mice were diminished: 0.3 μM in plasma and 0.4/1.2/0.8 μM in nmols/g of liver/kidney/brain. Treatment with 20NHS PEG-htCBS C15S resulted in significant elevation of Cth in plasma (39 M) and kidney homogenate (36 μM in nmols/g of tissue) and lesser increase in liver and brain homogenates (5 and 3 μM in nmols/g of tissue) compared to the untreated 1278T mice (p<0.05 for both plasma and tissue homogenates). Although plasma and kidney levels of Cth were substantially higher in the treated 1278T mice in comparison to healthy heterozygous controls (1 μM in plasma, p<0.01 and 1.2 μM in nmols/g in kidney, p<0.01) likely due to activity of 20NHS PEG-htCBS C15S in circulation, they were not quite normalized to healthy levels in liver (20 μM in nmols/g of tissue, non-significant) and brain homogenates (8 μM in nmols/g of tissue, p<0.05).

No significant differences were observed in plasma and tissue levels of Met or GSH among the untreated 1278T mice, the 20NHS PEG-htCBS C15S-treated 1278T mice and healthy heterozygous controls.

Levels of thioethers homolathionine (Hlth) and lanthionine (Lth) in plasma and tissue homogenates have also been identified as emerging surrogate markers of H₂S biogenesis. The levels of Hlth were markedly elevated in plasma (143 nM) and liver/kidney/brain homogenates of the untreated 1278T mice (6351/3324/1065 nM in pmol/g of tissue) and the 20NHS PEG-htCBS C15S treatment resulted in substantial correction of these levels to 45 nM in plasma (non-significant) and 170/1317/520 nM in pmols/g of liver/kidney/brain tissue (p<0.05/0.05/0.01). Interestingly, while liver Hlth in healthy heterozygous mice was significantly higher (996 nM in pmol/g of tissue, p<0.01) compared to the treated 1278T mice, the treatment resulted just in partial reduction of Hlth levels in the remaining compartments compared to those found in heterozygous mice: 8 nM in plasma (p<0.05) and 247/22 nM in pmol/g of kidney/brain tissues (p<0.01/0.001).

Regarding the second thioether Lth, only the plasma levels were significantly elevated in treated 1278T compared to the untreated ones (75 versus 17 nM, p<0.01), while levels of Lth in tissue homogenates remained similar (21/113/110 versus 14/92/104 nM in pmols/g of liver/kidney/brain tissue) due to low tissue permeability or a lack of a specific transporter for Lth. Plasma Lth in healthy heterozygous mice was significantly lower (32 nM, p<0.01), while its tissue levels were either elevated in liver (122 nM in pmols/g of tissue, p<0.05) and brain (304 nM in pmols/g of tissue, p<0.001) or remained similar in kidney (145 nM in pmols/g of tissue) when compared to the levels observed in the treated 1278T mice. Therefore, administration of 20NHS PEG-htCBS C15S improved or restored metabolic balance in plasma and tissues of the treated 1278T mice.

Example 10. Comparing methylation capacity among KO, HO, and 1278T mice treated with 20NHS PEG-htCBS CBS

Plasma and Tissue Levels of the SAM/SAH Ratio in 1278T and KO Mice were compared to the ratio in healthy heterozygous control mice and untreated mice. The 1278T adolescent mice (n=3) were treated for a period of 3 weeks with PEG-htCBS C15S (3× a week, SC, 7.5 mg/kg) and compared to age-matched untreated 1278T mice (n=3) and untreated healthy heterozygous controls (n=3). Treated KO mice were dosed 3× a week 7.5 mg/kg from day 2 of age. Each group consisted of at least three 18 days old mice. All data were compared using multivariate ANOVA followed by Tukey's post hoc test to determine significance: * p<0.05, ** p<0.01, *** p<0.001, ns—non-significant.

Methylation capacity of the untreated 1278T mice was substantially decreased based on SAM/SAH ratio in plasma (0.4) and liver/kidney/brain homogenates (0.3/0.4/0.2) and was substantially ameliorated to a ratio of 3 in plasma (p<0.001) and 0.7/2.1/1.5 in liver/kidney/brain homogenates (non-significant for liver; but p<0.01 for both kidney and brain tissue) in the treated group. The improved methylation capacity of the 20NHS PEG-htCBS C15S-treated 1278T mice approached or even normalized the one determined in healthy heterozygous mouse plasmas (5, p<0.01) and liver/kidney/brain homogenates (1.3/3.9/2.0, non-significant for liver and brain, but p<0.05 for kidney).

Administration of 20NHS PEG-htCBS C15S was also observed to significantly improve the SAM/SAH ratio in plasma and tissues of the treated KO mice compared to the untreated ones but did not normalize it to the levels seen in healthy heterozygous controls. The SAM/SAH ratio was quite small in the livers of WT controls compared to plasma and other tissue levels and, more importantly, comparable to the levels found in KO mice (0.78 and 0.94 in control healthy heterozygous and KO, p=ns). In addition, the 20NHS PEG-htCBS C15S treatment further decreased the SAM/SAH ratio in the treated KO mice compared to untreated ones significantly (0.28, p<0.01). Therefore, administration of 20NHS PEG-htCBS C15S improved or restored the metabolic balance in plasma and tissues of the treated KO mice.

Example 11. Pharmacokinetics of 20NHS PEG-htCBS C15S in Rats

Pharmacokinetic properties of 20NHS PEG-htCBS C15S were also determined in wild-type Sprague Dawley rats. CBS specific activity in plasma of male (black, n=11 each group) and female rats (gray, n=8 each group) after a single SC administration of 8 mg/kg (solid lines) or 24 mg/kg 20NHS PEG-htCBS C15S (dashed lines) in log scale is shown in FIG. 9A.

The bioavailability of 20NHS PEG-htCBS C15S was observed to be nearly identical at the different doses (8 and 24 mg/kg) suggesting that the observed differences in absorption in rats were likely due to sexual dimorphism. The elimination phase of 20NHS PEG-htCBS C15S was log-linear in all cohorts and conformed to first order kinetics meaning that the time required for half the concentration of 20NHS PEG-htCBS C15S activity to clear from the plasma was constant. The mean value of elimination half-life across groups was 42±2 hours. Despite differences between male and female rats, the PK parameters estimated from plasma levels of 20NHS PEG-htCBS C15S activity after SC dosing were dose proportional to 24 mg/kg.

Differences due to sexual dimorphism in rats were also observed in a repeated dosing study, where the rats received a total of 9 injections 48 hours apart of 4, 8, and 24 mg/kg 20NHS PEG-htCBS C15S (FIG. 9B and FIG. 9C). In general, initial as well as steady state levels of 20NHS PEG-htCBS C15S activity in plasma were higher in female than male rats at the same dose level correlating with the observation for the single dose study. Plasma levels of 20 NHS PEG-htCBS C15S after repeated dosing demonstrated little or no accumulation at any dose level, even though the doses were administered when only slightly more than half of the 20NHS PEG-htCBS C15S have cleared from the plasma (48 hours dosing interval versus average t1/2-E=42 hours). To aid in the understanding of these unexpected observations, the approach to steady state and the predicted steady state plasma levels were calculated after hypothetical repeated dosing using the simulation function in the PK software. After multiple doses, the observed plasma levels of 20NHS PEG-htCBS C15S activity were 15-52% and 0-35% of the predicted values for the peaks and troughs at steady state, as shown in Table 8. (N/A=not applicable)

TABLE 8 Predicted and observed c_(max-SS) and c_(min-SS) Male (8 mg/kg) Female (8 mg/kg) Male (24 mg/kg) Female (24 mg/kg) PK parameter Observed Predicted Observed Predicted Observed Predicted Observed Predicted c_(max) (mU/μl) 26 N/A 43 N/A 110 N/A 167 N/A c_(max-SS) (mU/μl) 10 66 42 127 99 227 196 378 c_(min-SS) (mU/μl) 0 41 3 61 60 171 78 249 Observed versus predicted 20NHS PEG-ht CBS C15S plasma activity at steady state (% or predicted value) c_(max-SS) 15 33 44 52 c_(min-SS) 0 5 35 31

The observed c_(max) and c_(min) correspond to the highest and lowest concentration of 20NHS PEG-htCBS C15S observed in plasma after the 2nd dose. The predicted values are from simulations using the time-concentration curves from the single dose PK study. The plasma levels after repeated dosing were observed to be closer to their predicted values at 24 mg/kg dose than at 8 mg/kg dose in both sexes.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a”, “an”, and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention. 

The invention claimed is:
 1. A method of PEGylating human truncated cystathionine β-synthase (CBS) protein containing a mutation of a cysteine to a serine at amino acid position 15 of SEQ ID NO: 1 (htCB C15S), the method comprising: (a) conjugating htCBS C15S with one or a plurality of NHS ester PEG molecules in solution at a molar excess of the NHS ester PEG molecules up to about 20-fold to create a batch, wherein the NHS ester PEG molecules are 5 kDa, 10 kDa, or 20 kDa NHS ester PEG molecules; (b) comparing a retention time from a chromatographic profile from Size Exclusion Chromatography-High Performance Liquid Chromatography (SEC-HPLC) analysis of the batch to a retention time from a chromatographic profile from SEC-HPLC analysis of a sample of NHS PEGylated htCBS C15S with acceptable PEGylation, wherein the sample of NHS PEGylated htCBS C15S with acceptable PEGylation contains htCBS C15S molecules with at least 5 PEGylated sites; and (c) if the batch has a retention time greater than a retention time of the sample of NHS PEGylated htCBS C15S with acceptable PEGylation, adding additional NHS ester PEG molecules to the batch to increase PEGylation of the htCBS C15S in the batch.
 2. The method of claim 1, wherein the plurality of NHS ester PEG molecules consists of 5 kDa NHS ester (5NHS) PEG molecules.
 3. The method of claim 1, wherein the plurality of NHS ester PEG molecules consists of 10 kDa NHS ester (10NHS) PEG molecules.
 4. The method of claim 1, wherein the plurality of NHS ester PEG molecules consists of 20 kDa NHS ester (20NHS) PEG molecules.
 5. The method of claim 1, wherein each of the plurality of NHS ester PEG molecules is less than about 20 kDa.
 6. The method of claim 1, wherein the retention time of the batch is greater than about 9.50 minutes.
 7. The method of claim 1, wherein the retention time of the batch is between about 9.60 minutes and about 9.70 minutes.
 8. The method of claim 1, wherein the retention time of the batch is within a range from about 9.50 to about 9.60 minutes.
 9. The method of claim 1, wherein the retention time of the sample of NHS PEGylated htCBS C15S with acceptable PEGylation is less than about 9.53 minutes.
 10. The method of claim 1, wherein the retention time of the batch is greater than about 9.75 minutes.
 11. The method of claim 1, wherein the retention time of the batch is greater than about 10.00 minutes.
 12. The method of claim 1, wherein the retention time of the batch is greater than about 10.25 minutes. 